DEVELOPMENTS IN GENETICS AND MOLECULAR BIOLOGY

MOLECULAR BIOLOGY & DIAGNOSTICS 311 Late 20th century…

INTRODUCTION TO MOLBIO AND DIAGNOSTICS
PRELIM: WEEK 1 LEC
o (amazing development in biotechnology have taken place)
- Human Genome Project
OBJECTIVES
- Cloning of organisms
1. Define appropriately Molecular Biology and Diagnostics
- Stem cell research
2. Discuss the development of Genetics
- Gene therapy
3. Discuss the overview of Molecular Biology
1866 Gregor Mendel published his work on Genetics. This
INTRODUCTION
suggesting the concepts of alleles and genes as discrete
MOLECULAR BIOLOGY
units of heredity
o JAMES WATSON – coined the term “molecular biology”
1869 Friedrisch Miescher is known as the first man to isolate
o A branch of biology dealing with the ultimate physicochemical
DNA (nucleic acids) He named it “nuclein”.
organization of living matter and especially with the molecular
1910 Morgan revealed that the units of heredity are contained
basis of inheritance and protein synthesis.
within chromosomes.
o The study of nucleic acids.
1944 Avery confirmed that it was the DNA that carried the
CLINICAL MOLECULAR LABORATORY genetic information through experimenting on bacteria.
o Molecular techniques are designed for the handling and analysis 1953 Franklin and Wilkins studied DNA by x-ray crystallography
of the nucleic acids, DNA and ribonucleic acid (RNA). which led to the unravelling of the double-helix structure
o The molecular biology laboratory, may be a separate entity or of DNA in 1953 by Watson and Crick.
part of an existing molecular diagnostics or molecular pathology 1960s Arber discovered restriction enzymes and later on, Smith
unit. demonstrated that DNA can be cleaved by restriction
o Nucleic acids offer several characteristics that support their use enzymes.
for clinical purposes. 1975 Southern blot has been invented
o Highly specific analyses can be carried out through hybridization 1977 DNA sequencing methods were developed, and the first
and amplification techniques complete DNA sequence of an organism, a
o (advantage: highly sensitive; it’s greatest weakness bacteriophage, was published.
1981 Prenatal genetic diagnosis of sickle cell disease was first
MOLECULAR DIAGNOSTICS shown to be feasible by Kan and Chang
o is referred to as the detection of genomic variants, aiming to 1985 Mullis and coworkers developed the Polymerase Chain
facilitate detection, diagnosis, subclassification, prognosis, and Reaction (PCR).
monitoring response to therapy 1996 DNA microarrays, which allow the simultaneous
o Application of laboratory medicine, genomics knowledge, and interrogation of gene transcripts were developed
technology in the field of molecular genetics. 2001 Draft of the human genome was released.
o Identification and fine characterization of the genetic basis of
inherited diseases which is vital for the accurate provision of a THE HUMAN GENOME PROJECT
physician’s diagnosis. o Started in October 1990.
o The HGP generated the first sequence of the human genome –
DIAGNOSTIC MOLECULAR BIOLOGY provided fundamental information about the human blueprint,
o Fastest growing area of laboratory medicine. which has since accelerated the study of human biology and
o Potential for a major impact on clinical medicine within the next improved the practice of medicine
decade. o April 2003 - The Human Genome Project is completed.
o It relates how structural variations in DNA and RNA are
associated with the development of chronic diseases.
o Nucleic acids also give valuable info with regards to the
pathology of a patient.
o Effective prevention and treatment of disease will result from
the analysis of these sequences in the clinical laboratory

[SARANILLO, KA.]
 BIOSAFETY LEVEL 3 (BSL-3)
MOLECULAR BIOLOGY & DIAGNOSTICS 311 o lab coats, gloves, eye protection, face shields, respirators
BIOSAFETY AND WASTE MANAGEMENT o Applicable to: laboratories in which work is performed using indigenous
PRELIM: WEEK 2 LEC or exotic biological agents with a potential for respiratory transmission
LABORATORY BIOSAFETY and those that may cause serious and potentially lethal infection.
o (WHO) Describes containment principles, technologies and o Requires: As described in BSL-2
practices implemented to prevent unintentional exposure to BSC-III
pathogens and toxins, or their accidental release.
Standard microbiological practices:
o (CDC) The application of safety precautions that reduce a
o As described in BSL-2
laboratorian’s risk of exposure to a potentially infectious o Persons below 16 years of age are not permitted entry.
microbe and limit contamination of the work environment and,
ultimately, the community. Special practices:
o Objective:Containment of potentially hazardous biological o As described for BSL-2 (Laboratory doors are kept close when
agents and toxins. experiments are in progress.)
o Purpose: To reduce the risk of exposure to staff and the o Laboratory clothing that protects street clothes is to be worn in the
unintentional release of hazardous biological agents or toxins laboratory, removed when exiting the laboratory, and decontaminated
prior to laundry or disposal.
into the surrounding community and environment.)
o Molded surgical masks or respirators are worn in rooms containing
experimental animals.
4 BIOSAFETY LEVELS o If animals housed with conventional caging system, personnel must wear
o (Center for Disease Control and Prevention and WHO) protective devices that includes wrap-around gowns, head covers,
gloves, shoe covers, and respirators; personnel shall shower on exit from
BIOSAFETY LEVEL 1 (BSL-1) areas where these devices are required.
o lab coats, gloves, eye protection o Vacuum lines are protected with high efficiency particulate air (HEPA)
o Generally appropriate for: undergraduate and secondary filters and liquid disinfectant traps.
educational training and teaching laboratories and for other o Spills and accidents which result in potential exposure to modified
organisms are immediately reported to the Biological Safety Officer.
laboratories that work with viable biological agents not known
o Written records are to be maintained on appropriate medical evaluation,
to consistently cause disease in healthy adult humans. surveillance, and treatment provided.
o Microorganisms under BSL-3: Mycobacterium tuberculosis,
Standard microbiological practices: St. Louis encephalitis virus, and
o Access to the laboratory is limited or restricted at the discretion Coxiella burnetii.
of the owner Laboratory facilities:
o Work can be performed on an open lab bench or table o Separated from open areas within the building.
o Work surfaces are decontaminated once a day, all liquid and o Accessed through two sets of doors. (anterooms)
o Physical separation from from other laboratories or activities.
solid wastes are decontaminated before disposal.
o Double-door clothes change room with showers.
o Mouth pipetting is prohibited. o Airlock technology §Interior surfaces of walls, floors, and ceilings are
o Eating, drinking, smoking, or storing food in the refrigerators is water resistant for easy cleaning, should be capable of being sealed for
prohibited. decontaminating the area.
o Procedures are performed carefully to prevent formation of aerosols. o Access doors are self-closing.
o Good hygiene including washing hands and wearing protective o HEPA-filtered exhaust air from BSC-I, II is discharged directly to the
clothes is encouraged. outside or through the building exhaust system.

Special practices: BIOSAFETY LEVEL 4 (BSL-4)

o lab coats, gloves, eye protection, face shields, respirators
o Contaminated materials to be decontaminated at a site away
o Applicable to: laboratories working with dangerous and exotic biological
from the laboratory are transported in durable, leak-proof agents that pose a high individual risk of life-threatening disease that
containers with closed lids. may be transmitted via the aerosol route and for which there is no
o Microorganisms under BSL-1: Bacillus subtilis, available vaccine or therapy.
Naeglaria gruberi, o Requires: As described in BSL-2
E. coli (nonpathogenic) BSC-III

BIOSAFETY LEVEL 2 (BSL-2) Standard microbiological practices:

o As described in BSL-3
o lab coats, gloves, eye protection, face shields
o Applicable to: laboratories in which work is performed using a Special practices:
broad-spectrum of biological agents and toxins that are o As described for BSL-3
associated with causing disease in humans of varying severity. o Access to the facility is limited by means of secure locked doors.
o Requires: AUTOCLAVE for disinfection o Any other material to be removed from the facility are to be autoclaved
BSC-I/BSC-II or decontaminated before exiting the maximum containment laboratory
o Personnel enter and exit the facility only through clothing change and
Standard microbiological practices: shower rooms; shower every time they exit the facility.
o Street clothing is removed and kept in an outer changing room. Complete
o As described in BSL-1
laboratory clothing (may be disposable) is provided and to be used by all
o Access to the laboratory is restricted when work is being conducted.
personnel entering the facility. When exiting, the laboratory clothing is
removed in an inner changing room before proceeding to the shower
Special practices: area. The clothing is decontaminated prior to laundering or disposal.
o As described for BSL-1 o Supplies and material are brought into the facility through a double-door
o Limited access to the laboratory and established policies and autoclave, fumigation chamber, or airlock.
procedures where persons entering the laboratory are aware of
the hazard and meet any specific entry requirements (such as Laboratory facilities:
immunization). o maximum containment facility is to be housed in a separate building
o The hazard-warning sign with the universal biosafety symbol (with any o Internal surfaces of walls, floors, and ceilings of the facility should be
special requirements for entry) are to be posted on the access door. water, acid, and alkali resistant
o Benchtops have seamless surfaces impervious to acids, alkalis, organic
o Protective clothing used exclusively in the laboratory is required;
solvents, and moderate heat
gloves are to be used to prevent skin contamination with o Access doors are self-closing and locking.
experimental organisms. o The supply and exhaust airflow is monitored by manometers to assure
o Only needle-locking hypodermic syringes are used, placed in inward (or zero) airflow at all times.
puncture-proof containers after use, and decontaminated o Exhaust air from the facility is filtered through HEPA filters before
before disposal. discharge to the outside.
o A biosafety manual is prepared and adopted for safety of personnel. o A specially designed suit area may be provided in the facility entry into
o Microorganisms under BSL-2: Staphylococcus aureus, which is through an airlock fitted with airtight doors.
o A chemical shower is provided to decontaminate the surface of the suit
HBV, HIV,
before the worker exits the area.
Salmonella, Toxoplasma.

[SARANILLO, KA.]
 SAETY EQUIPMENT Table 3.2. Categories of segregated laboratory waste materials and
o PPE (Gown, goggles, gloves, mask, hair cap) their recommended treatment
o Heat-resistant (non-asbestos) gloves – for handling hot CATEGORY OF
glassware and dry ice LABORATORY WASTE TREATMENT
o Tongs – for handling hot beakers/ test tubes MATERIAL
o Spill kits – for acids, caustic materials, or flammable solvents Uncontaminated (non- Can be used or recycled or disposed of as
(should be located in convenient and appropriate sites in the infectious) material general municipal waste
laboratory) Contaminated sharps Must be collected in puncture-proof
(hypodermic needles, containers fitted with covers and treated as
CHEMICAL HYGIENE PLAN: scalpels, knives and infectious
broken glass)
1. Every laboratory must have a complete chemical inventory that
Contaminated material Must be first decontaminated (Chemically or
is updated annually.
for reuse or recycling physically) and then washed, thereafter it can
2. Material Safety Data Sheet should be readily accessible and be treated as uncontaminated (non-infectious
available to every employee 24 hours a day, 7 days a week. material)
Contaminated material Must be decontaminated onsite OR stored
EXPOSURE CONTROL PLAN for disposal safely before transportation to another site
o a plan that ensures the protection of laboratory workers against for decontamination and disposal
potential exposure to blood- borne pathogen. Contaminated material Must be incinerated onsite OR stored safely
o ensures that the medical wastes produced by the laboratory are for incineration before transportation to another site for
incineration
managed and handled in a safe and effective manner.
Liquid waste (including Should be decontaminated before disposal in
potentially the sanitary sewer
(TIETZ) CLASSIFICATION ACC. TO THE DEGREE OF EXPOSURE: contaminated liquids)
GROUP I ALL employees have occupational exposure to for disposal in the
blood or other potentially infectious materials sanitary sewer system
GROUP II SOME employees have occ. exposure.
(Instrument technician)
GROUP III do NOT have ANY occupational exposure to CLASSIFICATION OF PROPER WASTE DISPOSAL (COLOR CODING)
blood/other potentially infectious materials. BLACK general waste; non-infectious and dry (E.g. paper,
boxes, plastic)
STANDARD PRECAUTIONS GREEN general waste; non-infectious and wet (E.g. leftover
o All specimens are potentially infectious, so they should all be food, food wrapper)
handled with standard precautions using proper personal YELLOW infectious, pathological, and pharmaceutical wastes
protective equipment (PPE) to prevent disease transmission. (E.g. blood and body fluids, tissue specimens, used
PPEs, discarded objects in sharps container
TRANSMISSION- BASED PRECAUTIONS ORANGE radioactive wastes kept in leaded container (handled
o including respirators are used with airborne or contact- separately)
transmissible agents.

CONTACT PRECAUTIONS
o are designed for direct patient care where there is the potential
for direct exposure to infectious agents on or from the patient.

IN GENERAL...
o standard precautions including gloves and gowns as PPE are used
by the molecular laboratory technologist who has no direct con-
tact with patients.

EYE PROTECTION OR MASKS

o are required in cases where frozen tissue is being processed or
where spraying or splashing of sample may occur.

GLOVES
o are absolutely required for handling of RNA
o highly recommended not only as part of standard precautions
but also to protect nucleic acids from nuclease degradation

DECONTAMINATION AND WASTE MANAGEMENT

o Any surface or material known to be, or could potentially be,
contaminated by biological agents during laboratory operations
must be correctly managed to control biological risks.

DECONTAMINATION
o (WHO) Reduction of viable biological agents or other hazardous
materials on a surface or object(s) to a pre-defined level by
chemical and/or physical means

WASTE MANAGEMENT
o refers to the various schemes to manage and dispose of wastes.
It can be by discarding, destroying, processing, recycling, reusing,
or controlling wastes. The prime objective of waste management
is to reduce the amount of unusable materials and to avert
potential health and environmental hazards.

[SARANILLO, KA.]
 NUCLEOTIDES
MOLECULAR BIOLOGY & DIAGNOSTICS 311 o If the ribose sugar is phosphorylated, the molecule is a
REVIEW OF DNA & RNA STRUCTURE, MUTATIONS
PRELIM: WEEK 3 LEC
nucleoside mono-, di-, or triphosphate or a nucleotide. For
example, adenosine with one phosphate is adenosine
DEOXYRIBONUCLEIC ACID
monophosphate (AMP). Adenosine with three phosphates is
o A nucleotide polymer in which of the monomers contains
adenosine triphosphate (ATP)
deoxyribose, a
phosphate group, and
one of the heterocyclic/
nitrogenous bases
adenine, cytosine,
guanine, or thymine.
o The four nucleotide
building blocks of DNA
are molecules of about
700 kd
o Each nucleotide consists
of a five-carbon sugar,
the first carbon of which
is covalently joined to a nitrogen base and the fifth carbon to a
triphosphate moiety

Important characteristics of the nucleotide formation process of

adding a phosphate group to a nucleoside are the following:
1. The phosphate group is attached to the sugar at the C-5’ position
through a phosphate-ester linkage (phosphate diester bond)
2. As with nucleoside formation, a molecule of water is produced
in nucleotide formation. Thus, overall, two molecules of water
are produced in combining a sugar, base, and phosphate into a
nucleotide.

NUCLEOSIDES
o A nitrogen base bound to an unphosphorylated sugar is a
nucleoside
o It is a two-subunit molecule in which a pentose sugar is bonded
to a nitrogen-containing heterocyclic base
o Adenosine (A), guanosine (G), cytidine (C), and thymidine (T) are
nucleosides. DNA
BASE ABBREV. NUCLEOSIDE NUCLEOTIDE
Important characteristics of the nucleoside formation process of Adenine A Deoxyadenosine dAMP
combining two molecules: Guanine G Deoxyguanosine dGMP
1. The base is always attached to C-1’, which is always in a Cytosine C Deoxycytidine dCMP
ßconfiguration. For purine bases, attachment is through N-9; for Thymine T Deoxythymidine Dtmp
pyrimidine bases, N-1 is involved. The bond connecting the sugar
and base is a ß –N-glycosidic linkage. RNA
2. A molecule of water is formed as the two molecules bond BASE ABBREV. NUCLEOSIDE NUCLEOTIDE
together; a condensation reaction occurs. Adenine A Adenosine AMP
Guanine G Guanosime GMP
Cytosine C Cytidine CMP
Thymine U Uridine UMP

[SARANILLO, KA.]
 NUCLEIC ACIDS PRIMASE
o A nucleic acid chain grows by the o is a ribonucleic acid (RNA) synthesizing enzyme that lays down
attachment of the 5 phosphate short (6–11 bp) RNA primers required for priming DNA synthesis.
group of an incoming nucleotide o must work repeatedly on the lagging strand to prime synthesis
to the 3 hydroxyl group of the last of each Okazaki fragment.
nucleotide on the growing chain
o Addition of nucleotides in this way ELONGATION
gives the DNA chain a polarity; o Enzymes known as DNA polymerases are responsible creating
that is, it has a 5 phosphate end the new strand by a process called elongation
and a 3 hydroxyl end o DNA polymerase III binds to the strand at the site of the primer
o DNA found in nature is mostly and begins adding new base pairs complementary to the strand
doublestranded. during replication
o Because replication proceeds in the 5' to 3' direction on the
o We refer to DNA as oriented leading strand, the newly formed strand is continuous
in a 5 to 3 direction, and the o The lagging strand begins
linear sequence of the replication by binding with
nucleotides, by convention, multiple primers. Each
is read in that order. primer is only several bases
o Two strands exist in opposite apart
5 to 3/3 to 5 orientation held o DNA polymerase then adds
together by the hydrogen pieces of DNA, called
bonds between their respective bases (A with T and G with C). Okazaki fragments, to the
strand between primers. This process of replication is
discontinuous as the newly created fragments are disjointed

TERMINATION
o Once both the
o They are in antiparallel orientation with continuous and
the 5 end of one strand at the 3 end of discontinuous
the other Identical sequences will not strands are formed,
hybridize with each other. In later an enzyme called
sections we will appreciate the exonuclease
importance of this when designing removes all RNA
hybridization and amplification assays. primers from the
original strands and are replaced with appropriate bases
DNA REPLICATION o Another exonuclease “proofreads” the newly formed DNA to
o Semiconservative replication is the key to check, remove and replace any errors
maintaining the sequence of the nucleotides o Another enzyme called DNA ligase joins Okazaki fragments
in DNA through new generations. together forming a single unified strand. The ends of the linear
o The order of nucleotides is maintained because each strand of DNA present a problem as DNA polymerase can only add
the parent double helix is the template for a newly replicated nucleotides in the 5ʹ to 3ʹ direction
strand.
SUMMARY OF ENZYMES IN REPLIATION:
STEPS IN DNA REPLICATION HELICASE unwinds and separates double stranded DNA as it
1. Replication fork formation moves along the DNA. It forms the replication fork by
2. Primer binding breaking hydrogen bonds between nucleotide pairs
3. Elongation in DNA
4. Termination DNA PRIMASE a type of RNA polymerase that generates RNA
primers. Primers are short RNA molecules that act as
REPLICATION FORK FORMATION templates for the starting point of DNA replication
o Before DNA can be replicated, the DNA synthesize new DNA molecules by adding
POLYMERASE nucleotides to leading and lagging DNA strands
double stranded molecule must be
TOPOISOMERASE unwinds and rewinds DNA strands to prevent the
“unzipped” into two single strands OR DNA GYRASE DNA from becoming tangled or supercoiled
o In order to unwind DNA, these EXONUCLEASES group of enzymes that remove nucleotide bases
interactions between base pairs must be from the end of a DNA chain
broken. This is performed by an enzyme DNA LIGASE joins DNA fragments together by forming
known as DNA helicase phosphodiester bonds between nucleotides.
o DNA helicase disrupts the hydrogen
bonds between the pairs to separate the
strands into a Y shape known as the DNA MUTATIONS
replication fork. This area will be the o Mutation is a change in a DNA sequence
template for replication to begin o can result from DNA copying mistakes made during cell division,
o However, the replication fork is bi- exposure to ionizing radiation, exposure to chemicals called
directional; one strand is oriented in the 3' to 5' direction mutagens, or infection by viruses
(leading strand) while the other is oriented 5' to 3' (lagging o Mutations in somatic cells are non-transmissible may contribute
strand). The two sides are therefore replicated with two to change (malignant transformation).
different processes to accommodate the directional difference
TYPES OF DNA MUTATION
PRIMER BINDING
1. Point mutation/Substitution
o The leading strand is the simplest to replicate. Once the DNA
- Single base substitutions are called point mutations (a
strands have been separated
change in one nucleotide of the sequence
o a short piece of RNA called a primer binds to the 3' end of the
- TYPES: Missense
strand.
Silent
o The primer always binds as the starting point for replication
Non-sense mutations
o Primers are generated by the enzyme DNA primase
2. Frameshift v Deletions
- Insertions
- Duplication

[SARANILLO, KA.]
 MISSENSE MUTATION DUPLICATION MUTATION
o Type of mutation is a change in one DNA base pair that results in o consists of a piece of DNA
the substitution of one amino acid another in the protein made that is abnormally copied
by the gene one or more times. This
type of mutation may alter
the function of the
resulting protein.

DNA RECOMBINATION
o Occurs through the molecular process of crossing over or
physical exchange between molecules. A recombinant molecule
or organism is one that holds a new combination of DNA
sequences.
NONSENSE MUTATION
CONJUGATION
o also a change in one DNA base pair. Instead of substituting one
o F factor is known as an extrachromosomal circle of dsDNA with
amino acid for another, however, the altered DNA sequence
genes for coding
prematurely signals the cell to stop building a protein. This type
o Sexes: F+ and F
of mutation results in a shortened protein that may function
o Fertility factor: F+
improperly or not at all
o Involves transfer of fertility factor from F+ to F

SILENT MUTATIONS TRANSDUCTION

o Does not cause any change in the amino acid o Transduction: transfer of gene from virus to bacterium
o Silent mutation codes for the same amino acid. o Bacteriophage: type of virus that can infect bacterium

FRAMESHIFT MUTATIONS TRANSFORMATION

o Type of mutation occurs when the addition or loss of DNA bases o Involves gene transfer which requires the uptake of a naked DNA
changes a gene's reading frame. A reading frame consists of or a foreign gene to a host organism
groups of 3 bases (CODON) that each code for one amino acid o Plasmids - Carry genetic information. Due to their size and effect
o Shifts the grouping of these bases and changes the code for on the host cell, plasmids can carry only a limited amount of
amino acids. The resulting protein is usually nonfunctional. information. The plasmid DNA duplex is compacted, or
Insertions, deletions, and duplications can all be frameshift supercoiled
mutations

DELETION MUTATIONS
o Changes the number of DNA bases by removing a piece of DNA
o Small deletions may remove one or a few base pairs within a
gene, while larger deletions can remove an entire gene or several
neighboring genes
o The deleted DNA may alter the function of the resulting protein

INSERTION MUTATIONS
o Changes the number of DNA bases in a gene by adding a piece of
DNA. As a result, the protein made by the gene may not function
properly

[SARANILLO, KA.]
 RIBONUCLEIC ACID (RNA) o RNA synthesis proceeds along the DNA template until the
o It differs from DNA in the sugar moieties, having ribose instead polyadenylation signal is encountered. At this point the process
of deoxyribose and, in one nitrogen base component, having of termination of transcription is activated.
uracil instead of thymine
o RNA is synthesized as a single strand rather than as a double helix
o RNA species fold and loop upon themselves to take on as much
of a doublestranded character as possible
o RNA can also pair with complementary single strands of DNA or
RNA and form a double helix.

o The antisense strand of the DNA template has a sequence

identical to that of the RNA product (except for the U for T
substitution in RNA), but it does not serve as the template for the
RNA

TYPES/STRUCTURES OF RNA
RIBOSOMAL RNA
o rRNA is an important structural and functional part of the
ribosomes, cellular organelles where proteins are synthesized
o largest component of cellular RNA

MESSENGER RNA
o is the initial connection between the information stored in DNA
and the translation apparatus that will ultimately produce the
protein products responsible for the phenotype dictated by the
chromosome.

TRANSFER RNA
o To transfer amino acids to match the correct mRNA codon.

Transcription

o Transcription is the copying of one strand of DNA into RNA by a

process similar to that of DNA replication.
o the nucleolus, is the location of ribosomal RNA synthesis
o The polymerization of RNA from a DNA template is catalyzed by
RNA polymerase.
o After binding to its start site in DNA, a specific sequence of bases
called the promoter, RNA polymerase and its supporting
accessory proteins synthesize RNA using the base sequence of
one strand of the double helix (the antisense strand) as a guide
o RNA synthesis does not require a primer.
o Upon initiation of RNA synthesis, the first ribonucleoside
triphosphate retains all of its phosphate groups as the RNA is
polymerized in the 5 to 3 direction.

[SARANILLO, KA.]
 MICROORGANISMS
MOLECULAR BIOLOGY & DIAGNOSTICS 311 o Some bacteria and fungi have tough cell walls that must be
DNA AND RNA ISOLATION TECHNIQUES AND DETECTION
PRELIM: WEEK 4 LEC
broken to allow the release of nucleic acid
o Enzyme products, e.g., lyzozyme or zymolyase, that digest cell
DNA ISOLATION
wall polymers are commercially available
o purpose of isolation/extraction is to release the nucleic acid from
o Cell walls can be broken mechanically by grinding or by
the cell for use in subsequent procedures
vigorously mixing with glass beads
o Ideally, the target nucleic acid should be free of contamination
o Gentler enzymatic methods are less likely to damage
with protein, carbohydrate, lipids, or other nucleic acid (DNA
chromosomal DNA and thus are preferred for methods involving
free of RNA or RNA free of DNA)
larger chromosomal targets as opposed to plasmid DNA
o The initial release of the cellular material is achieved by breaking
o Treatment with detergent (1% sodium dodecyl sulfate) and
the cell and nuclear membranes (cell lysis)
strong base (0.2 M NaOH) in the presence of Trisbase,
o Following lysis, the target material is purified, and then the
ethylenediaminetetraacetic acid (EDTA), and glucose can also
concentration and purity of the sample can be determined
break bacterial cell walls.
o First done by Friedrich Miescher in 1869
o Boiling in 8% sucrose, 8% Triton X-100 detergent, Tris buffer, and
o First done thru density gradient centrifugation
EDTA after lysozyme treatment releases DNA that can be
immediately precipitated with alcohol
STEPS IN DNA ISOLATION
o CELL LYSIS – Lysis of cell in which the nucleic acids will be extracted
TYPES OF DNA ISOLATION
o REMOVAL OF DEBRIS – Removing proteins, lipids, and other
contaminants from the nucleic acids o Organic isolation methods
o DNA PURIFICATION – Transferring the nucleic acids to water, or o Inorganic isolation methods
a buffer solution that will preserve them without interfering with o Solid-phase isolation
subsequent work o Crude lysis
- Proteolytic lysis of fixed material
PREPARING THE SAMPLES FOR DNA ISOLATION - Extraction with Chelating resin (Chelex)
o Nucleic acid is routinely isolated from human, fungal, bacterial,
and viral sources in the clinical laboratory ORGANIC ISOLATION METHODS
o TYPES OF SAMPLES: o After release of DNA from the cell, further purification requires
- Nucleated cells in suspensions removal of contaminating proteins, lipids, carbohydrates, and
- Tissue samples cell debris
- Microorganisms o uses a combination of high salt, low pH (acid), and an organic
mixture of phenol and chloroform (organic solvents)
NUCLEATED CELLS IN SUSPENSIONS o The combination readily dissolves hydrophobic contaminants
o The specimen may have to be pretreated to make nucleated cells such as lipids and lipoproteins, collects cell debris, and strips
available from which the nucleic acid will be extracted away most DNA-associated proteins
o White blood cells (WBCs) must be isolated from blood or bone o When phenol and chloroform are added to the hydrophilic
marrow specimens cleared cell lysate, a biphasic emulsion forms
- Differential density gradient centrifugation o Centrifugation will settle the hydrophobic layer on the bottom,
- Differential lysis with the hydrophilic layer on top. Lipids and other hydrophobic
components will dissolve in the lower hydrophobic phase; DNA
DENSITY GRADIENT CENTRIFUGATION will dissolve in the upper aqueous phase
o whole blood or bone marrow o The upper phase containing the DNA is collected, and the DNA is
mixed with isotonic saline is then precipitated using ethanol or isopropanol in a high
overlaid with Ficoll concentration of salt (ammonium, potassium or sodium acetate,
o Ficoll is a highly branched or lithium or sodium chloride).
sucrose polymer that does not o The DNA precipitate is collected by centrifugation. Excess salt is
penetrate biological membranes removed by rinsing the pellet in 70% ethanol, centrifuging and
o Upon centrifugation, the mononuclear WBCs (the desired cells discarding the ethanol supernatant, and then dissolving the DNA
for isolation of nucleic acid) settle into a layer in the Ficoll pellet in rehydration buffer, usually 10 mM Tris, 1 mM EDTA (TE),
gradient that is below the less dense plasma components and or water.
above the polymorphonuclear cells and red blood cells (RBCs)
o The layer containing the mononuclear cells is removed from the
tube and washed by at least two rounds of resuspension and
centrifugation in saline before proceeding with the nucleic acid
isolation procedure

DIFFERENTIAL LYSIS
o differences in the osmotic fragility of RBCs and WBCs
o Incubation of whole blood or bone marrow in hypotonic buffer
or water will result in the lysis of the RBCs before the WBCs
o The WBCs are then pelleted by centrifugation, leaving the empty
RBC membranes (ghosts) and hemoglobin, respectively, in INORGANIC ISOLATION METHODS
suspension and solution o Safety concerns in the clinical laboratory make the use of caustic
reagents such as phenol undesirable
TISSUE SAMPLES o Initially, these methods did not provide the efficient recovery of
o Fresh or frozen tissue samples must be dissociated before DNA clean DNA achieved with phenol extraction; however, newer
isolation procedures can be started. methods have proven to produce high quality DNA preparations
o Grinding the frozen tissue in liquid nitrogen, homogenizing the in good yields
tissue, or simply mincing the tissue using a scalpel can disrupt o Inorganic DNA extraction is sometimes called “salting out”. It
whole tissue samples makes use of low pH and high salt conditions to selectively
o Fixed embedded tissue has to be deparaffinized by soaking in precipitate proteins, leaving the DNA in solution.
xylene (dimethylbenzene) o The DNA can then be precipitated as described above using
o Less toxic xylene substitutes, such as Histosolve, Anatech Pro- isopropanol pelleted and re-suspended in TE buffer or water.
Par, or ParaClear, are also often used for this purpose
o After xylene treatment, the tissue is usually rehydrated by
soaking it in decreasing concentrations of ethanol

[SARANILLO, KA.]
 PREPARING SAMPLES OF RNA ISOLATION
o Preparation of specimen material for RNA extraction differs in
some respects than for DNA extraction
o Reticulocytes in blood and bone marrow samples:
- lysed by osmosis or separated from WBC’s by centrifugation
o Tissue samples:
- kept frozen in liquid nitrogen or immersed in buffer that will
inactivate intracellular RNAses (true for tissues such as
pancreas that contain large amounts of innate RNAses)
SOLID PHASE ISOLATION o Bacterial and fungal RNA:
o More rapid and comparably effective - chemical lysis or by grinding in liquid nitrogen
DNA extraction can be performed using o Viral RNA:
solid matrices to bind and wash the DNA - can be isolated directly from serum or other cell-free fluids
o Silica-based products were shown to by means of specially formulated spin columns or beads
effectively bind DNA in high salt
conditions ORGANIC ISOLATION METHODS
o Columns used in the clinical laboratory are usually small “spin o Called as Guanidine/Guanidium
columns” that fit inside microcentrifuge tubes. thiocyanate-phenol-chloroform
- These columns are commonly used to isolate viral and nucleic acid extraction
bacterial DNA from serum, plasma, or cerebrospinal fluid. o The cell lysis step for RNA
- They are also used routinely for isolation of cellular DNA in isolation is done in detergent or
genetics and oncology phenol in the presence of high
o Specific buffers are used to lyse bacterial, fungal, or animal cells. salt (0.2–0.5 M NaCl) or RNAse
Buffer systems designed for specific applications (e.g., bacterial inhibitors
cell lysis or human cell lysis) are commercially available o Guanidine thiocyanate is a strong denaturant of RNAses and can
o The cell lysate is applied to a column in high salt buffer, and the be used instead of high salt buffers
DNA in solution adsorbs to the solid matrix o Strong reducing agents such as 2-mercaptoethanol can also be
o After the immobilized DNA is washed with buffer, the DNA is added during this step
eluted in a specific volume of water, TE, or other low salt buffer o Once the cells are lysed, proteins can be extracted with phenol;
o The washing solutions and the eluant/eluent can be drawn Acid phenol:chloroform:isoamylalcohol (25:24:1) solution
through the column by gravity, vacuum, or centrifugal force. efficiently extracts RNA
o Chloroform enhances the extraction of the nucleic acid by
denaturing proteins and promoting phase separation (biphasic
emulsion)
o For RNA, the organic phase must be acidic (ph 4–5). The acidity
of the organic phase can be adjusted by overlaying it with buffer
of the appropriate Ph
o After phase separation, the upper aqueous phase containing the
RNA is removed to a clean tube, and the RNA is precipitated by
addition of two volumes of ethanol or one volume of isopropanol
o The RNA precipitate is then washed in 70% ethanol and
resuspended in RNF buffer or water.

SOLID PHASE EXTRACTION

o Solid-phase separation of RNA begins with similar steps as
described above for organic extraction
o The strong denaturing buffer conditions must be adjusted before
application of the lysate to the column
o As with DNA columns, commercial reagents are supplied with
the columns to optimize RNA adsorption and washing on the
silica-based matrix
o The lysate is applied to a column in high-salt chaotropic buffer,
and the adsorbed RNA is washed with supplied buffers. DNAse
can be added directly to the adsorbed RNA on the column to
remove contaminating DNA.
o Washing solutions and the eluant can be drawn through the
column by gravity, vacuum, or centrifugal force
o Small columns of silica-based material that fit inside microfuge
RNA ISOLATION tubes (spin columns) are widely used for routine nucleic acid
o Working with RNA in the laboratory requires strict precautions isolation from all types of specimens. The eluted RNA is usually
to avoid sample RNA degradation of sufficient concentration and purity for direct use in most
o RNA is especially labile due to the presence of RNAses. These applications.
enzymes are small proteins that can renature, even after
autoclaving, and become active
o Unlike DNAses, RNAses must be eliminated or inactivated before
isolation of RNA
o They remain active at a wide range of temperatures (e.g., below
-20C and can renature even after heating)

[SARANILLO, KA.]
 ISOLATION OF polyA (MESSENGER) RNA
o approximately 80%–90% of total RNA is rRNA; Often the RNA
required for analysis is mRNA, accounting for only about 2.5%–
5% of the total RNA yield
o To enrich the yield of mRNA, especially rare transcripts,
protocols employing oligomers of thymine or uracil immobilized
on a matrix resin column or beads are often used
o After washing away residual RNA, polyA RNA is eluted by
washing the column with warmed, low-salt buffer containing
detergent. With this approach, approximately 30– 40 ng of
mRNA can be obtained from 1 g of total RNA.

DNA/RNA DETECTION
o Laboratory analysis of nucleic acids produces variable results,
depending on the quality and quantity of input material
o This is an important consideration in the clinical laboratory, as
test results must be accurately interpreted with respect to
disease pathology
o METHODS:
- Electrophoresis
- Spectrophotometry
- Fluorometry
- NanoDrop
- Hybridization-based techniques

ELECTROPHORESIS
o DNA and RNA can be analyzed for quality by resolving an aliquot
of the isolated sample on an agarose gel (DNA/RNA)
o Fluorescent dyes:
- DNA visualization: Ethidium bromide, SybrGreen I
- RNA visualization: Ethidium bromide, SybrGreen II
o Silver stain has been used to detect small amounts of DNA by
visual inspection
o based on reduction of silver nitrate; is more sensitive than EtBr for
dsDNA and ssDNA or ssRNA with good sensitivity (picogram level)
o based on the reduction of silver cations to insoluble silver metal
by nucleic acids
o The appearance of DNA on agarose gels depends on the type of
DNA isolated. A good preparation of plasmid DNA will yield a
bright, moderate-mobility single band of supercoiled plasmid
DNA with minor or no other bands that represent nicked or
broken plasmid
o High-molecular-weight genomic DNA: bright band with low
mobility
o High-quality preparation of RNA: two distinct bands of Rrna
o If these bands are degraded (smeared) or absent, the quality of
the RNA in the sample is deemed unacceptable for use in
molecular assays.
o When fluorescent dyes are used, DNA and, less accurately, RNA
can be quantitated by comparison of the fluorescence intensity
of the sample aliquot run on the gel with that of a known amount
of control DNA or RNA loaded on the same gel

[SARANILLO, KA.]
 SPECTROPHOTOMETRY FLUOROMETRY
o Nucleic acids absorb light at 260 nm through the adenine o Fluorometry or fluorescent spectroscopy measures fluorescence
residues related to DNA concentration in association with DNA-specific
o Using the Beer-Lambert Law, concentration can be determined fluorescent dyes
from the absorptivity constants (50 for DNA, 40 for RNA). The o 3,5-diaminobenzoic acid 2HCl (DABA)
relationship of concentration to absorbance is expressed as: - Combines with alpha methylene aldehydes (deoxyribose) to
yield a fluorescent product
o Hoechst 33258
- combines with adenine-thymine base pairs in the minor
groove of the DNA double helix and is thus specific for intact
o The absorbance at this wavelength is thus directly proportional double-stranded DNA
to the concentration of the nucleic acid in the sample

DETERMINING CONCENTRATION OF NUCLEIC ACID

o To determine concentration, multiply the spectrophotometer
reading in absorbance units by the appropriate conversion factor
(50 ug/mL of DNA and 40 ug/mL of RNA)
o Most DNA and RNA preparations are of sufficient concentration
to require dilution before spectrophotometry
o If the sample has been diluted before reading, the dilution factor
must be included in the calculation of quantity

FORMULA:

EXAMPLES:
NANODROP
o Detection assays are persistently being developed that use
progressively smaller amounts of nucleic acid, often precluding
the use of conventional cuvette-based instruments for nucleic
acid quantitation for those that can perform micro-volume
quantitation
o This is a novel technology which allows usto measure nano-liter
volumes (pico concentration) of the nucleic acid (DNA or RNA)
sample.
o It is a type of spectrophotometer with a smaller sample size (as
much less as 1-2 microlitre) requirement and higher sensitivity
(even up to pico molar level). This is also a time saving
technology widely used in basic molecular biology research.

HYBRIDIZATION TECHNIQUES
o has higher resolution (down to the actual nucleotide sequence)
and utilizes "probe sequence" for DNA or RNA, and when it finds
its intended target, binds to it by hybridization process

[SARANILLO, KA.]
 o A smear located primarily in the lower region of the lane is a sign
MOLECULAR BIOLOGY & DIAGNOSTICS 311 that the isolated DNA is degraded. Either of these two latter
DNA HYBRIDIZATION TECHNIQUES
PRELIM: WEEK 5 LEC
conditions will prevent accurate analysis. If either is observed,
the DNA isolation and/or the restriction digest should be
HYBRIDIZATION TECHNIQUES
repeated accordingly.
o Procedures performed in the clinical molecular laboratory are
aimed at specific targets in genomic DNA
o This requires visualization or detection of a specific gene or
region of DNA in the background of all other genes
o There are several ways to find a particular region of DNA from
within an isolated DNA sample
o The initial method for molecular analysis of specific DNA sites
within a complex background was the Southern blot
o Modifications of the Southern blot are applied to analysis of RNA
and protein in order to study gene expression and regulation

SOUTHERN BLOT
o is named for Edwin Southern, who first reported the procedure PREPARATION OF RESOLVED DNA FOR BLOTTING (TRANSFER)
o In the Southern blot, DNA is isolated and cut with restriction o The resultant restriction fragments containing the target
sequence to be analyzed are obviously not distinguishable in the
enzymes
o Fragments are separated by gel electrophoresis, depurinated smear from other fragments that do not have the target
and denatured, and then transferred to a solid support such as sequence
nitrocellulose o To achieve optimal hydrogen bonding between the probe and its
o DNA fragments are exposed to a labeled probe (complementary complementary sequence in the resolved sample DNA, the
DNA or RNA) that is specific in sequence to the region of interest, double-stranded DNA fragments in the gel must be denatured
unbound probe is removed, and the signal of the probe is and transferred to a nitrocellulose membrane
detected to indicate the presence or absence (lack of signal) of
the sequence in question DEPURINATION
o As long as there is a probe of known identity, this procedure can o Before moving the DNA fragments from the gel to the membrane
analyze any gene or gene region in the genome at the molecular for blotting, the double-
level stranded DNA fragments must
be denatured, or separated, into
single strands.
o Although short fragments can be
denatured directly as described
below, larger fragments (500 bp)
are more efficiently denatured if
they are depurinated before
denaturation
o Therefore, for large fragments, the gel is first soaked in HCl
solution, a process that removes purine bases from the sugar
phosphate backbone. This will “loosen up” the larger fragments
for more complete denaturation

DENATURATION
o Following depurination, the DNA is denatured by exposing the
DNA in the gel to sodium hydroxide
o The strong base (NaOH) promotes breakage of the hydrogen
bonds holding the DNA strands to one another. The resulting
RESTRICTION ENZYME CUTTING AND RESOLUTION
single strands are then available to hydrogenbond with the
o After DNA isolation, the first step in the Southern blot procedure
single-stranded probe
is digestion of the DNA with restriction enzymes
o Further, the single-stranded DNA will bind more tightly than
o The choice of enzymes used will depend on the applications. For
double-stranded DNA to the nitrocellulose membrane upon
routine laboratory tests, restriction maps of the target DNA
transfer.
regions will have previously been determined, and the
appropriate enzymes will be recommended
BLOTTING (TRANSFER)
o 10 to 50 ug of genomic DNA are used for each restriction enzyme
o Before exposing the denatured sample DNA to the probe, the
digestion for Southern analysis
DNA must be transferred, or blotted, to a solid substrate that will
o After restriction enzyme digestion, the resulting fragments are
facilitate probe binding and signal detection
resolved by gel electrophoresis
o Substrate is usually nitrocellulose, nitrocellulose on an inert
o The percentage and nature of the gel will depend on the size of
support, nylon or cellulose modified with a diethyl amino ethyl,
the DNA region to be analyzed
or a carboxy methyl (CM) chemical group.
o As with all electrophoresis, a molecular weight standard should
o Membranes of another type, polyvinyl difluoride (PVDF), are
be run with the test samples. After electrophoresis, it is
used for immobilizing proteins for probing with antibodies
important to observe the cut DNA
(Western blots)
o The brightness of the DNA smears should be similar from lane to
lane, assuring that equal amounts of DNA were added to all
lanes. In any lane, a large aggregate of DNA near the top of the
gel indicates that the restriction enzyme activity was incomplete

[SARANILLO, KA.]
 MEMBRANE TYPES: ELECTROPHORETIC TRANSFER
o Single-stranded DNA avidly binds to nitrocellulose membranes o System utilizes electrodes attached to membranes above
with a noncovalent, but irreversible, connection. (anode) and below (cathode) the gel. The current carries the DNA
o The binding interaction is hydrophobic and electrostatic transversely from the gel to the membrane
between the negatively charged DNA and the positive charges o Carried out with a “tank” or by a “semidry” approach
on the membrane. o In the tank method, the electrodes transfer current through the
o Nitrocellulose-based membranes bind 70–150 ug of nucleic acid membrane through electrophoresis buffer
per square centimeter. o In the semidry method, the electrodes contact the gel-
o Membrane pore sizes (0.05 microns to 0.45 microns) are suitable membrane sandwich directly, requiring only enough buffer to
for small DNA fragments up to fragments 20,000 bp in length. soak the gel and membrane
o Membranes with a positive charge more effectively bind small o The tank electrophoretic transfer is preferred for large proteins
fragments of DNA. These membranes, however, are more likely resolved on acrylamide gels, whereas the semidry method is
to retain protein or other contaminants that will contribute to frequently used for small proteins.
background after the membrane is probed.
o Before transfer of the sample, membranes are moistened by
floating them on the surface of the transfer buffer.

PURE NITROCELLULOSE
o has a high binding capacity for proteins as well as nucleic acids.
o It is the most versatile medium for molecular transfer applications
o It is also compatible with different transfer buffers and detection
systems

REINFORCED
NITROCELLULOSE
o is more appropriate for applications where multiple probings
may be necessary
o Mechanically stable membranes can be formulated with a net
neutral charge to decrease the background. These membranes
have a very high binding capacity (400 ug/cm2), which increases VACUUM TRANSFER
sensitivity o Blotting technique uses suction to move the DNA from the gel to
the membrane in a recirculating buffer. Like electrophoretic
TRANSFER METHODS transfer, this method transfers the DNA more rapidly, e.g., in
1. CAPILLARY TRANSFER hours rather than days, than capillary transfer
2. ELECTROPHORETIC TRANSFER
3. VACUUM TRANSFER

CAPILLARY TRANSFER
o Gel is placed on top of a reservoir of buffer, which can be a
shallow container or membrane papers soaked in high salt
buffer, e.g., 10X saline sodium citrate (10X SSC: 1.5 M NaCl, 0.15
M Na citrate) or commercially available transfer buffers.
o Nitrocellulose membrane is placed directly on the gel, and dry
absorbent membranes or paper towels are stacked on top of the
membrane. The buffer is moved by capillary action from the
lower reservoir to the dry material on top of the gel.
o The movement of the buffer through the gel will carry the
denatured DNA out of the gel. When the DNA contacts the
nitrocellulose membrane, the DNA will bind to it while the buffer IMMOBILIZATION OF DNA AND PREHYBRIDIZATION
will pass through to the membranes or paper towels on top. o After binding the nucleic acid to membranes, the cut, denatured
DNA is permanently immobilized to the membrane by baking the
membrane in a vacuum oven (80C, 30–60 min.) or by UV cross-
linking, i.e., covalently attaching the DNA to the nitrocellulose
using UV light energy
o The purpose of baking or cross-linking is to prevent the
transferred DNA fragments from washing away or moving on the
membrane.
o Following immobilization of the DNA, a prehybridization step is
required to prevent the probe from binding to nonspecific sites
on the membrane surface, which will cause high background.
o Prehybridization involves incubating the membrane in the same
buffer in which the probe will subsequently be introduced. At
this point, the buffer does not contain probe.
o The buffer consists of blocking agents such as Denhardt solution
(Ficoll, polyvinyl pyrrolidane, bovine serum albumin) and salmon
sperm DNA. Sodium dodecyl sulfate (SDS, 0.01%) may also be
included, along with formamide, the latter especially for RNA
probes
o The membrane is exposed to the prehybridization buffer at the
optimal hybridization temperature for 30 minutes to several
hours, depending on the specific protocol. At this stage the
sample is READY FOR HYBRIDIZATION WITH THE PROBE, which
will allow visualization of the specific gene or region of interest

[SARANILLO, KA.]
 PROBES DETECTION SYSTEMS
o Purpose of the probe is to identify one or more sequences of o detect whether the probe has bound to the target molecule and,
interest within a large amount of nucleic acid. The probe if it has bound, the relative location of the binding
therefore should hybridize specifically with the target DNA or o After hybridization, unbound probe is washed off, and the blot is
RNA that is to be analyzed exposed to lightsensitive film to detect the fragments that are
o Can be RNA, denatured DNA, or other modified nucleic acids. hybridized to the radioactive/nonradioactive probe
Peptide nucleic acids (PNAs) and locked nucleic acids have also
been used as probes NON-RADIOACTIVE DETECTION
o These structures contain normal nitrogen bases that can o Non-radioactive detection systems require a more involved
hybridize with complementary DNA or RNA (target), but the detection procedure. For most nonradioactive systems, the
bases are connected by backbones different from the natural probe is labeled with a nucleotide covalently attached to either
phosphodiester backbone of DNA and RNA. These modified digoxygenin or biotin.
backbones are resistant to nuclease degradation and, because of o The labeled nucleotide is incorporated into the nucleotide chain
a reduced negative charge on their backbone, can hybridize of the probe by in vitro transcription, nick translation, primer
more readily to target DNA or RNA extension, or addition by terminal transferase
o Probes for Western blots are specific binding proteins or o Digoxygenin or biotin-labeled probe is incubated together with
antibodies. A labeled secondary antibody directed against the the blot with sample(s) containing the target sequence of
primary binding protein is then used for the visualization of the interest to allow for hybridization to occur
protein band of interest o After hybridization, unbound probe is washed away. Then,
antidigoxygenin antibody or streptavidin, respectively,
DNA PROBES conjugated to alkaline phosphatase (AP conjugate) is added to
o are created in several ways. A fragment of the gene to be reaction mix to bind to the digoxigenin- or biotin-labeled
analyzed can be cloned on a bacterial plasmid and then isolated probe:target complex
by restriction enzyme digestion and gel purification. The o After the binding of the conjugate, the membrane is bathed in a
fragment, after labeling and denaturation, can then be used in solution of substrate that, when oxidized by HRP or
Southern or Northern blot procedures dephosphorylated by AP, produces a signal. Substrates
o Other sources of DNA probes include the isolation of a sequence frequently used are dioxetane or tetrazolium dye derivatives,
of interest from viral genomes and in vitro organic synthesis of a which generate chemiluminescent or chromogenic signals,
piece of nucleic acid that has a particular sequence respectively
o The length of the probe will, in part, DETERMINE THE SPECIFICITY
OF THE HYBRIDIZATION REACTION
o Probe lengths range from tens to thousands of base pairs. The
probe is CONSTRUCTED SO THAT IT HAS A COMPLEMENTARY
SEQUENCE TO THE TARGETED GENE
o In order to bind to the probe then, the target nucleic acid has to
contain the sequence of interest. There are typically fewer
copies of a specific sequence in the genome, and therefore only
a few bands will be apparent after detection

RNA PROBES
o are often made by transcription (mRNA) from a synthetic DNA
template in vitro. These probes are similar to DNA probes with
equal or greater binding affinity to homologous sequences.
o Because RNA and DNA form a stronger helix than DNA/DNA, the
RNA probes may offer more sensitivity than DNA probes in the
Southern blot
o RNA probes can be synthesized directly from a plasmid template
or from template DNA produced by PCR. Predesigned systems
are commercially available for this purpose
o RNA probes are generally less stable than DNA probes and
cannot be stored for long periods. Synthesis of an RNA probe by
transcription from a stored template is relatively simple and RADIOACTIVE DETECTION
should be performed within a few days of use. o The chemiluminescent signal produced by the action of the
enzyme on dioxetane develops in the dark by autoradiography
PROBE LABELLING o For chromogenic detection, a colored signal is produced when
o In order to visualize the probe the enzyme interacts with a derivative of a tetrazolium dye and
bound to target fragments on is detected directly on the membrane filter
the blot, the probe must be
labeled and generate a INTERPRETATION OF RESULTS
detectable signal o When a specific probe binds to its target immobilized on a
o The original Southern membrane, the binding is detected as described in the previous
analyses used radioactive section, with the end result being the visual visualization of a
labeling with 32P. This “band” on the membrane or film
labeling was achieved by o A band is simply seen as a line running across the width of the
introduction of nucleotides lane. Analysis of bands, i.e., presence or absence or location in
containing radioactive the lane, produced by Southern blot can be straightforward or
phosphorus to the probe complex, depending on the sample and the design of the
o Today, many clinical procedure
laboratories use
nonradioactive labeling to
avoid the hazard and expense of working with radiation.
o Nonradioactive labeling methods are based on indirect detection
of a tagged nucleotide incorporated in or added to the probe.
The two most commonly used nonradioactive tags are biotin and
digoxygenin

[SARANILLO, KA.]
SUMMARY OF STEPS
1. DNA ISOLATION/EXTRACTION AND AMPLIFICATION.
2. RESTRICTION ENZYMES/DNA CUTTING
3. GEL ELECTROPHORESIS
4. TRANSFER/BLOTTING INTO NITROCELLULOSE MEMBRANE
5. IMMOBILIZATION OF TRANSFERRED DNA
6. PRE-HYBRIDIZATION
7. DNA HYBRIDIZATION OF LABELLED DNA PROBES
8. DETECTION
9. INTERPRETATION

OTHER HYBRIDIZATION TECHNIQUES

1. NORTHERN BLOT (RNA)
2. WESTERN BLOT (PROTEINS)
3. DOT BLOT
4. IN-SITU HYBRIDIZATION
5. FLUORESCENT IN-SITU HYBRIDIZATION (FISH)

[SARANILLO, KA.]
MOLECULAR BIOLOGY & DIAGNOSTICS 311
POLYMERASE CHAIN REACTION (PCR)
MIDTERM: WEEK 7 LEC

NUCLEIC ACID AMPLIFICATION

o Early analyses of nucleic acids were limited by the availability of
material to be analyzed
o Generating enough copies of a single gene sequence required
propagation of millions of cells in culture or isolation of large
amounts of genomic DNA
o The advent of the ability to amplify a specific DNA sequence
opened the possibility to analyze at the nucleotide level virtually
any piece of DNA in nature
o The first specific amplification method of any type was the
polymerase chain reaction (PCR)
o Other amplification methods have been developed based on
making modifications of PCR. The methods that have been
developed to amplify nucleic acids can be divided into three
groups, based on whether the target nucleic acid itself, a probe
specific for the target sequence, or the signal used to detect the
target nucleic acid is amplified

TARGET AMPLICATION
o Amplification of nucleic acids by target amplification involves
making copies of a target sequence to such a level (in the millions
of copies) that they can be detected in vitro
o This is analogous to growing cells in culture and allowing the cells
to replicate their nucleic acid as well as themselves so that, for
example, they can be visualized on an agar plate PCR PROFILE
o Polymerase chain reaction (PCR) is the first and prototypical 10 uL Mastermix 5 uL iTaq polymerase, Free nucleotides
method for amplifying target nucleic acid.
(dNTP’s) (super mix)
0.5 uL Forward Primer
POLYMERASE CHAIN REACTION (PCR)
0.5 uL Reverse Primer
o Kary Mullis in 1983 3.5 uL Distilled water
o The first successful amplification was a short fragment of the
0.5 uL DNA template
Escherichia coli plasmid, pBR322
Denaturation 95C for 30 seconds
o He called the method a “polymerase-catalyzed chain reaction”
Annealing 55C for 30 seconds
because DNA polymerase was the enzyme he used to drive the
Elongation 70C for 1 minute
replication of DNA, and once it started the replication continued
in a chain reaction Bacterial Cycle 28 times
o Since PCR was conceived and first performed, it has become Duration 1 hour and 30 minutes
increasingly user-friendly, more automated, and more amenable
to use in a clinical laboratory, with infinite applications possible DENATURATION
o Double-stranded DNA
BASIC PCR PROCEDURE is denatured into two
o When the cell replicates its DNA it requires the existing double- single strands in order
stranded DNA that serves as the template to give the order of to be replicated
the nucleotide bases, the deoxyribonucleotide bases o This is accomplished
themselves: adenine, thymine, cytosine, and guanine by heating the sample
o DNA polymerase to catalyze the addition of nucleotides to the at 94–96C for several
growing strand, and a primer to which DNA polymerase adds seconds to several
subsequent bases minutes, depending
o PCR essentially duplicates the in vivo replication of DNA in vitro, on the template
using the same components to replicate DNA as the cell does in o The initial denaturation step is lengthened for genomic or other
vivo, with the same end result, one copy of double-stranded DNA large DNA template fragments. Subsequent denaturations can
becoming two copies be shorter.
o Within one to two hours PCR can produce millions of copies
called amplicons of DNA; The real advantage of the PCR is the ANNEALING
ability to amplify specific targets o most critical step for the specificity of the PCR
o PCR presents the opportunity to amplify and essentially clone o In the second step of
the target sequences. The amplified target, then, can be the PCR cycle, the two
subjected to innumerable analytical procedures oligonucleotides that
o The components of the PCR, DNA template, primers, nucleotides will prime the
(dNTP’s), polymerase, and buffers, are subjected to an synthesis of DNA
amplification program anneal (hybridize) to
o Amplification program consists of a specified number of cycles complementary
that are divided into steps during which the samples are held at sequences on the
particular temperatures for designated times. The temperature template
will then determine the reaction that occurs, and changing the o The primers dictate
temperature changes the reaction. the part of the template that will be amplified; in other words,
the primers determine the specificity of the amplification
o It is important that the annealing temperature be optimized with
the primers and reaction conditions
o Annealing temperatures will range 50–70C and are usually
established empirically

[SARANILLO, KA.]
EXTENSION DNA POLYMERASE
o The third and last step of the PCR cycle is the primer extension o Automation of the PCR procedure was greatly facilitated by the
step; it is when DNA synthesis occurs discovery of the thermostable enzyme, Taq polymerase
o In this step, the polymerase synthesizes a copy of the template o The Taq polymerase was isolated from the thermophilic
DNA by adding bacterium, Thermus aquaticus
nucleotides to the o Using an enzyme derived from a thermophilic bacterium meant
hybridized primers. that the DNA polymerase could be added once at the beginning
DNA polymerase of the procedure and it would maintain its activity throughout
catalyzes the the heating and cooling cycles
formation of the o Tth polymerase
phosphodiester bond - From Thermus thermophilus, were subsequently exploited
between an incoming for laboratory use.
dNTP determined by - Tth polymerase also has reverse transcriptase activity so
hydrogen bonding to that it can be used in reverse transcriptase PCR (RT-PCR)
the template (A:T or
G:C) and the base at the 3′ end of the primer DEOXYRIBONUCLEOTIDE TRIPHOSPHATES (DNTP)
o In this way, DNA polymerase replicates the template DNA by o Nucleotide triphosphates are the building blocks of DNA
simultaneously extending the primers on both strands of the template. o An equimolar mixture of the four deoxynucleotide triphosphates
o This step occurs at the optimal temperature of the enzyme, 68–72C (dNTPs), adenine (dATP), thymine (dTTP), guanine (dGTP), and
o At the end of the three steps, or one cycle (denaturation, primer cytosine (dCTP), is added to the synthesis reaction in
annealing, and primer extension), one copy of double-stranded concentrations sufficient to support the exponential increase of
DNA has been replicated into two copies copies of the template
o Increasing the temperature back up to the denaturing o Standard procedures require 0.1–0.5 mM concentrations of each
temperature starts another cycle, with the end result being a nucleotide.
doubling in the number of double stranded DNA molecules again
o At the end of the PCR program, millions of copies of the original PRIMERS
region defined by the primer sequences will have been o are the critical component of the PCR because primers
generated determine the specificity of the PCR
o Are DNA oligonucleotides; single-stranded DNA (ssDNA)
fragments of usually 20–30 bases in length
o Are analogous to the
probes in blotting and
hybridization procedures.
Primers are chemically
manufactured on a DNA
synthesizer
o Primers are designed to
contain sequences
homologous to sites
flanking the region to be
analyzed
o Forward primer must bind to the target DNA sequence just 5′ to
the sequences intended to be amplified
o Reverse primer must bind just 5′ to the sequence to be amplified
on the opposite strand of the DNA.
o Thus, the design of primers requires some knowledge of the
target sequence. The placement of the primers will also dictate
the size of the amplified product.
o Factors that affect primer binding:
- Primer sequence (% GC)
PCR COMPONENTS
- Length
o The PCR is a method of in-vitro DNA synthesis. Therefore, to perform
PCR, all of the components necessary for the replication of DNA in
- Approximate melting temperature (Tm)
vivo are combined in optimal concentrations for replication of DNA o primer Tm can serve as a starting point for setting
to occur in vitro the optimal annealing temperature
o Components: o Primers should be designed such that the forward
- DNA template and reverse primers have similar Tm so that both
- Polymerase enzyme (DNA polymerase); w/ reverse will hybridize optimally at the same annealing
transcriptase for RT-PCR temperature
- Primers
o Tm can be adjusted by increasing the length of the
- Deoxynucleoside triphosphates (dNTP’s)
- buffer components including monovalent and divalent cations
primers or by placing the primers in areas with
more or fewer Gs and Cs in the template
DNA TEMPLATE
o May be single- or double-stranded DNA MISPRIMING
o In a clinical sample, depending on the application, the template o A fragment synthesized from mispriming will carry the primer
may be derived from: sequence and become a target for subsequent rounds of
- The patient’s genomic or mitochondrial DNA amplification.
- Viruses, Bacteria, Fungi, or Parasites o Misprimed products will consume components away from the
o For routine clinical analysis, 100 ng to 1 ug of DNA is usually used. intended reaction
Lesser amounts are required for more defined template o The resulting misprimed products may also interfere with proper
preparations such as cloned target DNA or product from a interpretation of results or subsequent procedures such as
previous amplification sequencing or mutation analysis
o Characteristics of a good DNA template:
- Free of contaminating proteins
- without nicks or breaks that can stop DNA synthesis or
cause misincorporation of nucleotide bases
o Templates with high GC content and secondary structure may
prove more difficult to optimize for amplification

[SARANILLO, KA.]
 TRIS BUFFER
o Tris buffer and accessory buffer components are also important
for optimal enzyme activity and accurate amplification of the
intended product
o 10 mM Tris-HCl maintains the proper pH of the buffer, usually
between pH 8 and pH 9.5.

BOVINE SERUN ALBUNIN (BSA)

o Bovine serum albumin (10–100 g/mL) binds inhibitors and
stabilizes the enzyme

DITHIOTHREITOL (DTT)
PRIMER-DIMERS o Dithiothreitol (0.01 mM) provides reducing conditions that may
o Secondary structure (internal folding and hybridization within enhance enzyme activity
DNA strands) can also interfere with PCR
o These are PCR products that are just double the size of the FORMAMIDE
primers. They result from the binding of primers onto each other o Formamide (1%–10%) added to the reaction mixture will lower
through short (2–3 base) homologies at their 3′ ends and the the denaturing temperature of DNA with high secondary
copying of each primer sequence. structure, thereby increasing the availability for primer binding.
o The resulting doublet is then a very efficient target for
subsequent amplification CHAOTROPIC AGENTS
o Chaotropic agents such as triton X-100, glycerol, and dimethyl
sulfoxide added at concentrations of 1%–10% may also reduce
secondary structure to allow polymerase extension through
difficult areas.

MASTER MIX
o Enzymes are usually supplied with buffers optimized by the
manufacturer.
o Commercial PCR buffer enhancers of proprietary composition
may also be purchased to optimize difficult reactions
o Often, the buffer and its ingredients are mixed with the
nucleotide bases and stored as aliquots of a master mix. The
PCR BUFFER enzyme, target, and primers are then added when necessary.
o provide the optimal conditions for enzyme activity o Dedicated master mixes will also include the primers, so that
- Potassium chloride, ammonium sulfate, and other salts only the target sequences must be added
of monovalent cations
- Magnesium chloride THERMAL CYCLER
- Tris buffer o Thermal cyclers or thermocyclers were thus designed to rapidly
- Bovine serum albumin and automatically ramp (change) through the required
- Dithiothreitol incubation temperatures, holding at each one for designated
- Formamide periods
- Chaotropic agents o There are numerous manufacturers of thermal cyclers. These
instruments differ in heating and/or refrigeration systems as well
KCl, AMMONIUM SULFATE, MONOCOVALENT CATIONS as the programmable software within the units
o Potassium chloride (20–100 mM), ammonium sulfate (15–30 o Samples may be held in open chambers for air heating and
mM), or other salts of monovalent cations are important buffer cooling or in sample blocks designed to accommodate 0.2-mL
components tubes, usually in a 96 well format
o These salts affect the denaturing and annealing temperatures of o Rapid PCR systems are designed to work with very small sample
the DNA and the enzyme activity volumes in chambers that can be heated and cooled quickly by
o An increase in salt concentration makes longer DNA products changing the air temperature surrounding the samples.
denature more slowly than shorter DNA products during the o Real-time PCR systems are equipped with fluorescent detectors
amplification process, so shorter molecules will be amplified to measure PCR product as the reaction proceeds.
preferentially. The influence of buffer/salt conditions varies with
different primers and templates

MAGNESIUM CHLORIDE (MgCl2)

o Magnesium chloride also affects primer annealing and is very
important for enzyme activity. Magnesium requirements will
vary with each reaction, because each NTP will take up one
magnesium atom
o Magnesium ions functions as a co-factor for activity of DNA
polymerases by enabling incorporation of dNTP’s during
polymerization PCR PROCEDURE
o The magnesium ions at the enzyme’s active site catalyze o For routine PCR in the laboratory, an appropriate amount of DNA
phosphodiester bond formation between the 3’-OH of a primer that has been isolated from a test specimen is mixed with the
and the phosphate group of the dNTP other PCR components, either separately or as part of a master
o The recommended range of MgCl2 concentration is 1–4 mM, in mix in 0.2–0.5–mL tubes
standard reaction conditions. o Most thermal cyclers take thin-walled tubes, 0.2 mL tube strips
o Too few Mg2 ions lower enzyme efficiency, resulting in a low or 96 well plates
yield of PCR product o Sample tubes are then loaded into the thermal cycler. The
o Overly high Mg2 concentrations promote misincorporation and computer is programmed with the temperatures and times for
thus increase the yield of nonspecific products each step of the PCR cycle, the number of cycles to complete
(usually 30–50), the conditions for ramping from step to step,
and the temperature at which to hold the tubes once all of the
cycles are complete.

[SARANILLO, KA.]
 VISUALIZATION OF PCR AMPLICONS
o After the PCR, a variety of methods are used to analyze the PCR
product. Most commonly, the PCR product is analyzed by gel or
capillary electrophoresis. Depending on the application, the size,
presence, or intensity of PCR products is observed on the gel

CONTROLS FOR PCR

o As with any diagnostic assay, running the correct controls in PCR
is essential for maintaining and ensuring the accuracy of the
assay. With every PCR run, the appropriate controls must be
included
o Positive controls ensure that the enzyme is active, the buffer is
optimal, the primers are priming the right sequences, and the
thermal cycler is cycling appropriately.
o Negative control without DNA (also called a contamination
control or reagent blank) ensures that the reaction mix is not
contaminated with template DNA or amplified products from a
previous run
o Negative control with DNA that lacks the target sequence
(negative template control) ensures that the primers are not
annealing to unintended sequences of DNA.
o In some applications of PCR, an internal control is included
o In this type of control (amplification control), a second set of
primers and an unrelated target are added to the reaction mix to
demonstrate that the reaction is working even if the test sample
is not amplified
o Amplification controls are performed, preferably in the same
tube with the test reaction, although it is acceptable to perform
the amplification control on a duplicate sample.
o This type of control is most important when PCR results are
reported as positive or negative, by which “negative” means that
the target sequences are not present.
o The amplification control is critical to distinguish between a true
negative for the sample and an amplification failure (false-
negative)

[SARANILLO, KA.]
 NESTED PCR
MOLECULAR BIOLOGY & DIAGNOSTICS 311 o The low level of target and the presence of interfering sequences
PCR MODIFICATIONS
MIDTERM: WEEK 8 LEC
can prevent a regular PCR from working with the reliability
required for clinical applications
PCR MODIFICATIONS
o Nested PCR is a modification that increases the sensitivity and
o PCR today has been adapted for various applications; several
specificity of the reaction
modifications are used in the clinical laboratory
o In nested PCR, two pairs of primers are used to amplify a single
o The methods that have been developed to amplify nucleic acids
target in two separate PCR runs. The second pair of primers,
can be divided into three groups:
designed to bind slightly inside of the binding sites of the first
- Target amplification
pair, will amplify the product of the first PCR in a second round
- Probe amplification
of amplification. The second amplification will specifically
- Signal amplification
increase the amount of the intended product
TARGET AMPLICATIONS
o Capable of detecting multiple targets in a single run (multiplex PCR)
REAL-TIME/QUANTITATIVE PCR (RT-PCR or qPCR)
o Using RNA templates (reverse transcriptase PCR) o Standard PCR procedures
o Amplified products as templates (nested PCR) will indicate if a particular
o Quantitating starting template (quantitative PCR, or real-time PCR) target sequence is present
(qualitative) in a clinical
MULTIPLEX PCR sample. For some
o More than one primer pair can be added to a PCR so that situations, though, the
multiple amplifications are primed simultaneously, resulting in clinician is also interested
formation of multiple products in how much of the target
o Multiplex PCR is especially useful in typing or identification analyses sequence is present
o Individual organisms, from viruses to humans, can be identified (quantitative)
or typed by observing a set of several PCR products at once o In contrast to real-time PCR, analysis of PCR product by the
o Pathogen typing and forensic identification kits contain multiple sets standard method occurs at the end of the PCR stationary phase
of primers that amplify polymorphic DNA regions. The pattern of (endpoint analysis)
product sizes will be specific for a given type or individual o Initially performed by adding ethidium bromide (EtBr) to a
o Multiple organisms have been the target of multiplex PCR in regular PCR. Because EtBr intercalates into double stranded DNA
clinical microbiology laboratories and fluoresces, it can be used to monitor the accumulation of
o One respiratory sample, for example, can be used to test for the PCR products during the PCR in real time as it is made.
presence of more than one respiratory virus o Advantage of this method over standard PCR is the ability to
o Organisms that cause sexually transmitted diseases can be determine the amount of starting template accurately
targeted in multiplex PCR using one genital swab
o In a slightly different approach to testing for multiple targets,
one set of primers can detect an infectious organism, and a
second set can detect the presence of a gene that makes that
organism resistant to a particular antimicrobial agent
o Multiplexing primers is useful, not only to detect multiple targets
but also to confirm accurate detection of a single target
o Internal amplification controls are often multiplexed with test
reactions that are interpreted by the presence or absence of product
o Internal amplification controls are the ideal for positive/negative
qualitative PCR tests

REVERSE TRANSCRIPTASE PCR

o Amplification by PCR requires a double-stranded DNA template;
If the starting material for a procedure is RNA, it must first be
converted to double-stranded DNA
o This is accomplished through the action of reverse transcriptase
(RT), an enzyme isolated from RNA viruses
o The enzyme first copies the RNA single strand into a RNA:DNA
hybrid strand and then uses a hairpin formation on the end of PRINCIPLE OF RT-PCR/qPCR
the newly synthesized DNA strand to prime synthesis of the o If the copy number is measured by detectable fluorescence, the
homologous DNA strand, replacing the original RNA in the hybrid curve looks similar to a bacterial growth curve, with a lag phase,
o The resulting double-stranded DNA is called cDNA for copy or an exponential (log) phase, a linear phase, and a stationary phase
complementary DNA. This product is adequate for PCR. o Exhaustion of reaction components and competition between
o Specific primers, oligo dT primers or random hexamers, are most PCR product and primers during the annealing step slow the PCR
often used to prime the synthesis of the initial DNA strand. product accumulation after the exponential phase of growth
Specific primers will prime cDNA synthesis only from transcripts until it finally plateaus
complementary to the primer sequences
o USES OF RT-PCR:
- measure RNA expression profiles
- detect Rrna
- analyze gene regions interrupted by long introns
- detect microorganisms with RNA genomes
o The detection of RNA viruses such as Coronavirus, which is
responsible for severe acute respiratory syndrome, can be
accomplished using RT PCR
o RT PCR was originally performed in two steps: cDNA synthesis and PCR
o Tth DNA polymerase, which has RT activity and proprietary mixtures
of RT and sequestered (hot-start) DNA polymerase, are components
of one-step RT PCR procedures.
o These methods are more convenient than the two-step procedure,
as RNA is added directly to the PCR
o The amplification program is modified to include an initial incubation
of 45–50C for 30– 60 minutes, during which RT makes cDNA from
RNA in the sample. The RT activity will then be inactivated in the first
denaturation step of the PCR procedure

[SARANILLO, KA.]
o Using the fluorescent signal to detect the growing target copy o A probe composed of a single-stranded DNA oligomer
number during the amplification process, analysis in real-time homologous to a specific sequence in the targeted region of the
PCR is performed in the exponential phase of growth where the PCR template is used. Note that this probe is present in the PCR
accumulation of fluorescence is inversely proportional to the in addition to the specific primers used to prime the DNA
amount of starting template synthesis reaction
o With 10-fold dilutions of known positive standards, a o The probe is
relationship between the starting target copy number and the chemically modified
cycle number at which fluorescence crosses a threshold amount at its 3′end so that it
of fluorescence can be established cannot be extended
o The PCR cycle at which sample fluorescence crosses the by the polymerase.
threshold is the threshold cycle (CT) The single-stranded
o Once this relationship is established, the starting amount of an DNA TaqMan probe
unknown specimen can be determined by the cycle number at is covalently
which the unknown crosses the fluorescence threshold attached to a
fluorescent dye on
one end and another
dye or non-
fluorescent
molecule (quencher) that pulls fluorescent energy from the 5′
dye on the other
o As the polymerase proceeds to synthesize DNA from the
template to which the probe is hybridized, the natural
exonuclease activity of Taq polymerase will degrade the probe
into single and oligonucleotides, thereby removing the labeled
nucleotide from the vicinity of the quencher and allowing it to
fluoresce. Excess probe is present so that with every doubling of
the target sequences more probe binds and is digested, and
more fluorescence is generated.

RT-PCR LABELLED PROBES

o More specific systems have been devised that utilize probes
designed to generate fluorescence
o Probes are fluorescently labelled DNA oligonucleotides which
are designed to bind downstream of one of the primers during
the PCR reaction and to give a fluorescent signal during the MOLECULAR BEACONS
reaction o Measures the accumulation of product at the annealing step in
o The probes increase specificity by only yielding fluorescence the PCR cycle.
when they hybridize to the target sequences o Signals are detectable only when the probes are bound to the
o EXAMPLES OF LABELLED PROBES: template before displacement by the polymerase
- TaqMan o The probe is chemically modified so that it is not degraded during
- Molecular beacons the extension step. Molecular beacons are designed with a ~25-
- FRET probes b–specific binding sequence flanked by a short (~5 b) inverted
repeat that will form a stem and loop structure (hairpin
TaqMan Assay structure) when the probe is not bound to the template. There
o TaqMan was developed is a reporter fluorophor (dye) at the 5′ end of the oligomer and
from one of the first quencher at the 3′ end
probe based systems o Until specific product is present, the probe will form a hairpin
for RT-PCR structure that brings the fluorophore in proximity with the
o This method exploits quencher (Fig. 7-19). Fluorescence will occur on binding of the
the natural 5′ to 3′ probe to denatured template during the annealing step (Fig. 7-
exonuclease activity of 20)
Taq polymerase to generate signal.
o The TaqMan procedure measures fluorescent signal generated
by separation of reporter dye and quencher

[SARANILLO, KA.]
o When the primers are extended in the PCR, displacement of the
probe by Taq will restore the hairpin (nonfluorescent) structure.
Excess probe in the reaction mix will assure binding to the
increasing amount of target. The amount of fluorescence,
therefore, will be directly proportional to the amount of
template available for binding and inversely proportional to the
CT.

OTHER RT-PCR LABELLED PROBES:

- Eclipse probes
- Amplifluor assays
- Scorpions PCR primers
- LUX PCR primers
- QZyme PCR primers

ARBITRARILY PRIMED PCR

o In arbitrarily primed PCR, also known as randomly amplified
polymorphic DNA or random amplification of polymorphic DNA
(RAPD), short (10–15 bases) primers with random sequences are
used to amplify arbitrary regions in genomic DNA under low
stringency conditions
o PCR products are generated without knowing the sequence of
the target or targeting a specific gene. It is possible with this
method to obtain multiple products, depending how many times
a short sequence appears in the genome; in traditional PCR, only
one product is generally obtained
o Arbitrarily, primed PCR has been used primarily in the
FLUORESCENT RESONANCE ENERGY TRANSFER (FRET) epidemiological typing of microorganisms. Similar band patterns
o Utilizes two specific probes, one with a 3′ fluorophore (acceptor), obtained from performing PCR with the same arbitrary primers
the other with a 5′ catalyst for the fluorescence (donor) that indicate that two organisms are the same or similar.
binds to adjacent targets
o Examples of frequently used donor acceptor pairs are
fluorescein-rhodamine, fluorescein2-aminopurine, and
fluorescein-Cy5. When the donor and acceptor are brought
within 1– 10 nm (1–5 bases) through specific DNA binding,
excitation energy is TRANSFERRED from the donor to the
acceptor
o The acceptor then loses the energy in the form of heat or
fluorescence emission called sensitized emission. The sequences
of FRET probes are designed such that they bind 1–5 bases from
each other on the target sequences. When they are bound to the
target, the fluorescence is catalyzed. As with the molecular
beacons, the more template available for binding, the more
fluorescence will be generated.

[SARANILLO, KA.]
 TRANSCRIPTION-BASED AMPLIFICATION SYSTEMS
o RNA is the usual target instead of DNA. A DNA copy (cDNA) is
synthesized from the target RNA, and then transcription of the
DNA produces millions of copies of RNA
o STEPS:
- A primer complementary to sequences in the target RNA
that also has the binding site for RNA polymerase at one end
is added to a sample of target RNA. The primer anneals, and
reverse transcriptase makes a DNA copy of the target RNA
- Heat is used to denature the DNA/RNA hybrid, and a second
primer binds to the cDNA and is extended by reverse
transcriptase producing double-stranded DNA. RNA
polymerase derived from the bacteriophage T7 then
transcribes the cDNA, producing hundreds to thousands of
copies of RNA. The transcribed RNA can then serve as target
RNA to which the primers bind and synthesize more cDNA.
o TYPES:
- Transcription-mediated amplification (TMA)
- Nucleic acid sequence–based amplification (NASBA)
- Self-sustaining sequence replication (3SR)

OTHER AMPLIFICATION METHODS

PROBE AMPLIFICATION
o In probe amplification procedures, the number of target nucleic
acid sequences in a sample is NOT CHANGED as it is in target
amplification procedures like PCR
o Synthetic probes that are specific to the target sequences bind
to the target where the probes themselves are amplified
o Types:
- Ligase chain reaction (LCR; Abbott)
- Strand displacement amplification (SDA; Becton Dickinson)
- QB replicase (Vysis)

SIGNAL AMPLIFICATION
o Signal amplification procedures differ from target amplification
procedures in that the number of target sequences does not
change
o Large amounts of signal are bound to the target sequences that
are present in the sample
o Because the number of target sequences does not change, signal
amplification procedures are inherently better at quantitating
the amount of target sequences present in the clinical sample
o Types:
- Branched DNA amplification
- Hybrid Capture assays
- Cleavage-based amplification

[SARANILLO, KA.]
 TYPE II RESTRICTION ENZYMES
MOLECULAR BIOLOGY & DIAGNOSTICS 311 o Are those used most frequently in the laboratory. These enzymes
RESTRICTION ENZYMES AND RESTRICTION ENZYME MAPPING
MIDTERM: WEEK 10 LEC
do not have inherent methylation activity in the same molecule
as the nuclease activity. They bind as simple dimers to their
RESTRICTION ENZYMES
symmetrical DNA recognition sites
o Are DNA-cutting enzymes
o These sites are palindromic in nature; that is, they read the same
o Each enzyme recognizes one
5 to 3 on both strands of the DNA (Fig. 1-13), referred to as
or a few target sequences and
bilateral symmetry. Type II restriction enzymes cleave the DNA
cuts DNA at or near those
directly at their binding site, producing fragments of predictable size
sequences
o Type II restriction enzymes have been found in almost all
o Endonucleases break the
prokaryotes, but none, to date, have been found in eukaryotes
sugar phosphate backbone of
DNA at internal sites; these
enzyme that cleaves DNA into
fragments at or near specific
recognition sites within
molecules known as
restriction sites
o Restriction enzymes are endonucleases that recognize specific
base sequences and break or restrict the DNA polymer at the
sugar-phosphate backbone. These enzymes were originally
isolated from bacteria where they function as part of a primitive
defense system to cleave foreign DNA entering the bacterial cell.
The ability of the cell to recognize foreign DNA depended on both OVERHANGS
DNA sequence recognition and methylation o Some enzymes cut the duplex with a staggered separation at the
o Genetic engineering was stimulated by the discovery of recognition site, leaving 2–4 base single-strand overhangs at the
deoxyriboendonucleases, or endonucleases ends of the DNA
o Restriction enzymes are named for the organism from which o The single-strand ends CAN HYBRIDIZE WITH COMPLEMENTARY ENDS on
they were isolated other DNA fragments, directing the efficient joining of cut ends
o EXAMPLES: o Because of their ability to form hydrogen bonds with
- BamHI was isolated from Bacillus amyloliquefaciens H complementary overhangs, these cuts are said to produce
- HindIII from Haemophilus influenzae Rd “sticky ends” at the cut site
- SmaI from Serratia marcescens Sbb

USES OF RESTRICTION ENZYMES

o The specificity of their action and the hundreds of enzymes
available that recognize numerous sites are key factors in the
ability to perform DNA recombination in vitro:
- Genetic mapping
- DNA cloning
- Genetic engineering and genetic rearrangement
- Mutation detection and analysis

DNA RECOMBINATION

RESTRICTION ENZYMES GROUPS

o Natural restriction enzymes are arranged in five groups:
- Type I - Type IV
- Type II - Type V
- Type III

TYPE I RESTRICTION ENZYMES

o Have both nuclease and methylase activity in a single enzyme
o They bind to host-specific DNA sites of 4–6 bp separated by 6–8
bp and containing methylated adenines
o The site of cleavage of the DNA substrate can be over 1000 bp
from this binding site
o The distance from the recognition site makes type I restriction BLUNT
endonucleases less helpful in the field of genetic engineering o Another mode of cutting separates the DNA duplex at the same
o An example of a type I enzyme is EcoK from E.coli K 12. It place on both strands, leaving flush, or blunt, ends. These ends
recognizes the site: can be rejoined as well, although not as efficiently as sticky ends

o Where N represents nonspecific nucleotides and the adenine

residues (A) are methylated

[SARANILLO, KA.]
 TYPE III RESTRICTION ENZYMES
o Resemble type I enzymes in their ability to both methylate and
restrict (cut) DNA
o Like type I, they are complex enzymes with two subunits
o Rarely used in genetic engineering as they cut DNA sequences
well outside of the recognition sequence and need to detect two
separate sequences to achieve this. This means they are not
always able to provide complete restriction enzyme digestion
o An example of a type III enzyme is
HinfIII from H. influenzae. It
recognizes the site where the
adenine methylation occurs on only
one strand:
o Mapping of a circular plasmid is slightly different, as there are no
free ends (Fig. 6-3). The example shown in the figure is a 4-kb
TYPE IV RESTRICTION ENZYMES
pair circular plasmid with one BamHI site and two XhoI sites.
o Can only cleave methylated DNA (DNA that is not transcribed
Cutting the plasmid with BamHI will yield one fragment. The size
into a protein) and sequence specificity is weak
of the fragment is the size of the plasmid. Two fragments
released by XhoI indicate that there are two XhoI sites in the
TYPE V RESTRICTION ENZYMES
plasmid and that these sites are 1.2 and 2.8 kb pairs away from
o Require guide RNA (gRNA) to target specific sequences and it is
each other.
these that are being modified or used in genome engineering
o As with linear mapping, cutting the plasmid with XhoI and BamHI
methods such as TALENS and CRISPR-Cas9. CRISPR is the short
at the same time will start to order the sites with respect to one
form of clusters of regularly interspaced short palindromic
another on the plasmid. One possible arrangement is shown in
repeats CRISPR regions refer to repeated nucleotide and spacer
Figure 6-3. As more enzymes are used, the map becomes more
patterns within a section of the DNA; it is within spacers that
detailed
viruses incorporate their DNA. By inserting other genetic code
into a spacer by artificial means it is possible to modify the
genome of a living organism

RESTRICTION ENZYME MAPPING

o Clinical and forensic analyses require characterization of specific
genes or genomic regions at the molecular level
o Because of their sequence-specific activity restriction
endonucleases provide a convenient tool for molecular
characterization of DNA
o Restriction enzymes commonly used in the laboratory have four
to six base pair recognition sites, or binding/ cutting sites, on the
DNA.
o Restriction site mapping
- determining where in the DNA sequence a particular
restriction enzyme recognition site is located, was initially
developed using small circular bacterial plasmids. The
resultant maps were used to identify and characterize
naturally occurring plasmids and to engineer the o The pattern of fragments produced by restriction enzyme
construction of recombinant plasmids digestion can be used to identify that DNA and to monitor certain
o To make a restriction map, DNA is exposed to several restriction changes in the size, structure, or sequence of the DNA
enzymes separately and then in particular combinations. Take, o The location and order of restriction enzyme sites on a DNA
for example, a linear fragment of DNA cut with the enzyme PstI fragment is a molecular characteristic of that DNA. The resulting
o After incubation with the enzyme, the resulting fragments are differences in the size or number of restriction fragments are
separated by gel electrophoresis. The gel image reveals four called restriction fragment length polymorphisms (RFLPs)
fragments, labeled A, B, C, and D, produced by PstI (Fig. 6-1) o RFLPs were the basis of the first molecular-based human
o From the number of fragments one can deduce the number of identification and mapping methods
PstI sites: three. The sizes of the fragments, as determined by o RFLPs can also be used for the clinical analysis of structural
comparison with known molecular-weight standards, indicate changes in chromosomes associated with disease
the distance between these sites or from the site to the end of (translocations, deletions, insertions, etc.)
the fragment.
o Although PstI analysis of this fragment yields a characteristic
four-band restriction pattern, it does not indicate the order of
the four restriction products in the original fragment. To begin to
determine the order of the restriction fragments, another
enzyme is used, for example BamHI. Cutting the same fragment
with BamHI yields two pieces, indicating one BamHI site in this
linear fragment (see Fig. 6-1)

[SARANILLO, KA.]
MOLECULAR BIOLOGY & DIAGNOSTICS 311
DNA SEQUENCING
MIDTERM: WEEK 11 LEC

DNA SEQUENCING
o The term DNA sequencing refers to sequencing methods for
determining the order of the nucleotide bases - adenine,
guanine, cytosine, and thymine - in a molecule of DNA
o Researchers still, however, need to be able to determine the
sequence of bases in DNA that make up the human genome. As
such, DNA sequencing has been developed but the process itself
is a seemingly complex one. DNA sequencing involves the
determination of the order of DNA bases
o In the clinical laboratory, DNA sequence information (the order
of nucleotides in the DNA molecule) is used routinely for a
variety of purposes:
- Detecting mutation
- Typing microorganisms
- Identifying human haplotypes
- designating polymorphisms
- targeted therapies will be directed at abnormal DNA
sequences detected by these techniques

HISTORY OF DNA SEQUENCING

o The sequencing of DNA molecules began in the
1970s with development of the Maxam-
Gilbert method, and later the Sanger method
o Originally developed by Frederick Sanger in
1975, most DNA sequencing that occurs in
medical and research laboratories today is
performed using sequencers employing
variations of the Sanger method

HOW DNA SEQUENCING IS PERFORMED

o DNA sequencing involves the process of figuring out the precise
order of the four bases found in one piece of DNA
o The DNA is really just a template that is used to create a series of
o The denaturing conditions (formamide, urea, and heat) prevent
fragments
the single strands of DNA from hydrogen bonding with one
o The fragments differ in length by one base and they are
another or folding up so that they migrate through the gel strictly
separated by size before the bases are identified, which then
according to their size
effectively recreates the original DNA sequence
o The migration speed is important because single-base resolution
o Each person has twenty-three pairs of chromosomes - one copy
is required to interpret the sequence properly.
of the human genome
o After electrophoresis, the gel apparatus is disassembled; the gel
o Because technology has limitations, we are limited in how many
is removed to a sheet of filter paper, and it is dried on a gel dryer.
bases can be read at one time
The dried gel is exposed to light sensitive film
o Therefore, we can't just read each base from one end of a
o The sequence is inferred from the bands on the film
chromosome to the other. To make it feasible, the chromosome
o The smallest (fastest-migrating) band represents the base
is cut down into smaller fragments
closest to the labeled end of the fragment. The lane in which that
band appears identifies the nucleotide.
MANUAL DNA SEQUENCING
o Sequence is read from the bottom (5′ end of the DNA molecule)
o Direct determination of the order, or sequence, of to the top (3′ end of the molecule) of the gel
nucleotides in a DNA polymer is the most specific and direct o INTERPRETATION:
method for identifying genetic lesions (mutations) or - G + A and G → Read as Guanine
polymorphisms, especially when looking for changes - (G) G + A → Read as Adenine (A)
affecting only one or two nucleotides - C + T and C → Read as Cytosine (C)
o TYPES: Maxam-Gilbert method - C + T → Read as Thymine (T)
Sanger method
PROCEDURE
MAXAM-GILBERT METHOD (CHEMICAL SEQUENCING) 1. Prepare DNA sample
o Maxam-Gilbert chemical sequencing method was developed in 2. Aliquot DNA into four test tubes and add different chemicals
the late 1970s by Allan M. Maxam and Walter Gilbert - Tube 1: Dimesthylsulphate
o Maxam-Gilbert sequencing requires a double- or single-stranded - Tube 2: Formic acid
version of the DNA region to be sequenced, with one end - Tube 3: Hydrazine
radioactively labeled - Tube 4: Hydrazine with salt
o For sequencing, the labeled fragment, or template, is aliquoted 3. Add hot piperadine at 90°C
into four tubes. Each aliquot is treated with a different chemical 4. Introduce the four test tubes in polyacrylamide gel and start the
with or without high salt electrophoresis and cover the gel by an x-ray film
o Upon addition of a strong reducing agent, such as 10% piperidine, - Well 1: Tube 1
the single-stranded DNA will break at specific nucleotides
- Well 2: Tube 2
o After the reactions, the piperidine is evaporated, and the
- Well 3: Tube 3
contents of each tube are dried and resuspended in formamide
- Well 4: Tube 4
for gel loading. The fragments are then separated by size on a
5. Read the sequence starting from the bottom
denaturing polyacrylamide gel

[SARANILLO, KA.]
 PROCEDURE:
1. 1:1 mixture of template and primer is placed into four separate
reaction tubes in sequencing buffer
2. Sequencing buffer is usually provided with the sequencing
enzyme and contains ingredients necessary for the polymerase
activity
3. Mixtures of all four dNTPs and one of the four ddNTPs (dNTP +
ddNTP) are then added to each tube, with a different ddNTP in
LIMITATIONS OF CHEMICAL SEQUENCING each of the four tubes
o Although Maxam-Gilbert sequencing is a relatively efficient way 4. With the addition of DNA polymerase enzyme to the four tubes,
to determine short runs of sequence data the reaction begins. After about 20 minutes, the reactions are
o The method is not practical for high throughput sequencing of terminated by addition of a stop buffer The stop buffer consists
long fragments. In addition, the hazardous chemicals hydrazine of 20 mM EDTA to chelate
and piperidine require more elaborate precautions for use and cations and stop enzyme activity,
storage. formamide to denature the
o This method has therefore been replaced by the dideoxy chain products of the synthesis
termination sequencing method for most sequencing reaction, and gel loading dyes
applications (bromophenol blue and/or
xylene cyanol)
DIDEOXY (SANGER) SEQUENCING 5. The sets of synthesized fragments are then loaded onto a
o Discovered by Frederick Sanger and colleagues in 1977; It was denaturing polyacrylamide gel
the widely used sequencing methods for 25 years after its 6. The products of each of the sequencing reactions are loaded into
discovery four adjacent lanes, labeled A, C, G, or T, corresponding to the
o The original dideoxy chain termination sequencing methods ddNTP in the four reaction tubes.
required a single-stranded template 7. Once the gel is dried and exposed to x-ray film, the fragment
o Templates up to a few thousand bases long could be produced patterns can be visualized from the signal on the 32P-labeled
using M13 bacteriophage, a bacterial virus with a single stranded primer or nucleotide
DNA genome - The four-lane gel electrophoresis pattern of the products of
o Dideoxy chain termination (Sanger) sequencing is a modification the four sequencing reactions is called a sequencing ladder
of the DNA replication process. A short, synthetic single- - The ladder is read to deduce the DNA sequence. From the
stranded DNA fragment (primer) complementary to sequences bottom of the gel, the smallest (fastest migrating) fragment
just 5′ to the region of DNA to be sequenced is used for priming is the one in which synthesis terminated closest to the
dideoxy sequencing reactions primer
o For detection of the products of the sequencing reaction, the
primer may be attached covalently at the 5′ end to a 32P-labeled
nucleotide or a fluorescent dye-labeled nucleotide

o Just as in the in vivo DNA replication reaction, an in vitro DNA

synthesis reaction would result in polymerization of
deoxynucleotides to make full-length copies of the DNA
template
o modified dideoxynucleotide (ddNTP) derivatives are added to
the reaction mixture
o Dideoxynucleotides lack the hydroxyl group found on the 3′
ribose carbon of the deoxynucleotides (dNTPs)
o DNA synthesis will stop upon incorporation of a ddNTP into the
growing DNA chain (chain termination) because without the
hydroxyl group at the 3′ sugar carbon, the 5′-3′ phosphodiester
bond cannot be established to incorporate a subsequent
nucleotide
o The newly synthesized chain will terminate with the ddNTP

[SARANILLO, KA.]
 AUTOMATED FLUORESCENT SEQUENCING
o uses double-stranded templates and cycle sequencing
o Because cycle sequencing (unlike manual sequencing) does not
require sequential addition of reagents to start and stop the
reaction, cycle sequencing is more easily adaptable to high
throughput applications and automation
o Electrophoresis and reading of the sequencing ladder can also be DYE TERMINATOR SEQUENCING
automated o is performed with one of the four fluorescent dyes attached to
- use of fluorescent dyes instead of radioactive nucleotides each of the ddNTPs instead of to the primer
to label the primers or sequencing fragments o Primer is unlabeled
- Fluorescent dyes used for sequencing have distinct “colors,” o Advantage of this approach is that all four sequencing reactions
or peak wavelengths of fluorescence emission, that can be are performed in the same tube (or well of a plate) instead of in
distinguished by automated sequencers four separate tubes
- advantage of having four distinct colors is that all four of the o After addition of the rest of the reaction components and cycle
reaction mixes can be read in the same lane of a gel or on a sequencing, the product fragments are labeled at the 3′ end. As
capillary with dye primer sequencing, the “color” of the dye corresponds
to the ddNTP that terminated the strand

APPROACHES IN AUTOMATED SEQUENCING

o Goal of both approaches is the same: to label the fragments
synthesized during the sequencing reaction according to their
terminal ddNTP
- Dye Primer sequencing
- Dye terminator sequencing

DYE PRIMER SEQUENCING

o Four different fluorescent dyes are attached to four separate
aliquots of the primer
o The dye molecules are attached covalently to the 5′ end of the
primer during chemical synthesis, resulting in four versions of
the same primer with different dye labels
o The primer labeled with each “color” is added to four separate
reaction tubes, one each with ddATP, ddCTP, ddGTP, or ddTTP
o After addition of the rest of the components of the sequencing
reaction and of a heat stable polymerase, the reaction is
subjected to cycle sequencing in a thermal cycler.
o The products of the sequencing reaction are then labeled at the
5′ end, the dye color associated with the ddNTP at the end of the
fragment

[SARANILLO, KA.]
 INDIRECT ELISA
MOLECULAR BIOLOGY & DIAGNOSTICS 311 o Antigen is immobilized
TECHNIQUES IN PROTEIN ANALYSIS
FINALS: WEEK 14 LEC
directly on the plate but
includes the addition of
PROTEINS
o are the products of transcription and translation of the nucleic acids
unconjugated (unlabeled)
o Even though nucleic acids are most often the focus of molecular antibody which will bind to
analysis, the ultimate effect of the information stored and the specific antigen then
delivered by the nucleic acid is manifested in proteins adds another conjugated
o Analysis of the amount and mutational status of specific proteins (labelled) antibody that
has long been performed in situ using immunohistochemistry, on directly binds to the
live cells using flow cytometry, and on isolated proteins by unconjugated antibody
Western blots o USES: measures endogenous antibodies
o More recently, global protein analysis by mass spectrometry o ADVANTAGES: Amplification using secondary antibody (labeled Ab)
(proteomics) has been applied to clinical work on a research basis o DISADVANTAGES: Potential for cross-reactivity caused by
o Even if proteins are not being tested directly, they manifest the secondary antibody
phenotype directed by the nucleic acid information

TECHNIQUES USED IN ANALYZING PROTEINS

o WESTERN BLOT
o ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
o IMMUNOHISTOCHEMISTRY
o FLOW CYTOMETRY
o PROTEOMICS
o MALDI-TOF

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA

o Biochemical assay that uses antibodies and an enzyme mediated SANDWICH ELISA
color change to detect the presence of biomolecules such as o Most common type of ELISA
proteins, peptides, hormones or antibodies present in the o Capture antibody is coated on the plate which captures the
sample target antigen
o Part of the principle under labeled immunoassays o Addition of conjugated antibody is then added to bind the
o TYPES OF ELISA: antigen that has been captured
- Direct elisa - Sandwich elisa o USES: determines the analyte concentration in the sample
- Indirect elisa - Competitive elisa o ADVANTAGES:
- Highest specificity and sensitivity
REAGENTS AND MATERIALS - Compatible with complex sample matrices
o Blocking buffer such as 5% bovine serum albumin dissolved in o DISADVANTAGES: Longer protocol and strenuous to do
phosphate buffer saline (PBS) that covers all unsaturated
surface binding sites in the microplate well
o Surfactant such as Tween-20 that is added to blocking and
wash buffer, helps to minimize hydrophobic interactions
between blocking protein and antigen or antibody
o Standard should have a known concentration of the target
protein or molecule
o Control should have a valid and known concentration of the
antigen that will be use to validate the ELISA procedure
o Horseradish peroxidase (HRP) and alkaline phosphatase is the
most common enzyme labeled used in labelled
immunoassays
o Beta galactosidase, acetylcholinesterase and catalase are also
considered as enzymes used in the labelled immunoassays

DIRECT ELISA
o Antigen is immobilized directly on the plate and uses a
conjugated antibody that can bind specific to the target protein COMPETITIVE ELISA
o USES: assessing antibody affinity and specificity; presence of o Capture antibody is coated on the plate but uses a conjugated
blocking or inhibitors antigen that will compete for the binding with the antigen
o ADVANTAGES: Fast and simple protocol present in the sample.
o DISADVANTAGES: o The more antigen in the sample, the less conjugated antigen will
- Less specific bind to the capture antibody
- Presence of high background if all proteins from sample are o USES: determines the concentration of small molecules and hormones
immobilized o ADVANTAGES: Ability to quantify small molecules
o DISADVANTAGES:
- Less specific
- Requires conjugated antigen

[SARANILLO, KA.]
 WESTERN BLOT/IMMUNOBLOT STAINS USED TO VISUALIZE PROTEINS
o A technique for detecting specific proteins separated by o Coosamie Brilliant Blue- most common (50
electrophoresis by the use of labeled antibodies ng protein band)
o An analytical technique used to detect proteins in a given sample o Silver Staining- more sensitive (up to 50
times more sensitive than CBB)
of tissue homogenate or extract
o Dependent on the quality of antibody used to probe the protein
of interest, and how specific the antibody is for the protein
C. TRANSFER / BLOTTING
o The membrane is placed on top
PRINCIPLE OF WESTERN BLOT
o an immunoblotting technique which rely on the specificity of binding
of the gel, and stack of filter
between a molecule of interest and a probe to allow detection a papers are placed on top of the
molecule of interest in mixture of many other similar molecules gel. The entire stack is placed in
o The molecule of interest is a protein and a probe is typically an a buffer solution which moves
antibody raised against that particular pattern up the paper by capillary action,
bringing the proteins with it
STEPS IN WESTERN BLOTTING o Examples of membrane
1. Extraction of protein support:
samples - Nitrocellulose paper
2. Resolution of the - Nylon paper
protein sample in - Polyvinylidene fluoride (PVDF)
sodium dodecyl
sulfate
polyacrylamide D. BLOCKING
denaturing gel o Blocking of the membrane by using non-specific proteins
electrophoresis o Blocking of non-specific binding is achieved by placing the
(SDS-PAGE) membrane in a dilute solution of protein (Ex. Bovine Serum
3. Transfer of the Albumin)
separated polypeptides to a membrane support o The protein in the dilute solution attaches to the proteins in all
4. Blocking nonspecific binding sites on the membrane places where the target proteins have not attached. Thus, when
5. Addition of antibodies antibody is added, there is no room on the membrane for it other
6. Detection using autoradiography than on the binding sites of the specific target protein
o Washing of the nitrocellulose paper with a wash solution (Tris-
A. PROTEIN EXTRACTION buffer saline + Tween 80
o physical and chemical based methods to disrupt the cell’s plasma
membrane and release the proteins inside it
o Sources of proteins:
- prokaryotic or - Viruses
eukaryotic cells - Environmental samples
- Tissues - Purified proteins
o Methods for protein extraction
- Chemical methods
- Physical methods

CHEMICAL METHODS
o Uses various types of detergents to make proteins accessible
o Weak detergents- used to extract water soluble proteins inside E. ADDITION OF ANTIBODIES
the cell w/o disrupting intracellular membrane compartments o The membrane is “probed” for the protein of interest with a
o Strong detergents- solubilize proteins found in all membranes primary antibody which will bind to target proteins
o EXAMPLE: Sodium Dodecyl Sulfate (SDS) o Addition of a secondary antibody which is a conjugated antibody
- is an anionic detergent; Denatures secondary and non– (Ex. Conjugated Horse radish peroxidase) will bind to the Fc
disulfide–linked tertiary structures region of the primary antibody
- used to impart a negative charge to linearized proteins o Horse radish perodixase - catalyzes a reaction that produces light
as a by-product. The amount of light produced is directly
PHYSICAL METHODS proportional to the amount of HRP-conjugated antibody bound
o Protein extraction is done at cold temperatures (usually 4C) to o Secondary antibodies are linked to a reporter
avoid protein denaturation enzyme/fluorophore, which when exposed to an appropriate
o EXAMPLES: substrate (Ex. luminol) drives a colorimetric reaction and
- Blender (for larger samples) produces a color
- Homogenizer (smaller
volumes)
- Sonicator

B. GEL ELECTROPHORESIS
o Proteins of the sample are separated using SDS-PAGE (Sodium
dodecyl polyacrylamide gel electrophoresis)
o SDS- applied to protein sample to linearize proteins and to
impart a negative charge to linearized proteins

F. DETECTION OF PROTEIN BANDS

o Either Colorimetric or
Chemiluminescent
o Viewed using
autoradiography (X-ray films)

[SARANILLO, KA.]
 CHROMOSOMAL DISORDER
MOLECULAR BIOLOGY & DIAGNOSTICS 311 o occurs when there is a change in the number or structure of the
DETECTION OF GENETIC DISORDERS
FINALS: WEEK 15 LEC
chromosomes
o This change in the amount, or arrangement of, the genetic
INTRODUCTION
information in the cells may result in problems in growth,
o Molecular and cytogenetic analyses are a critical component of
development and/or functioning of the body systems
diagnostic testing, especially for diseases that arise from known
o EXAMPLES:
genetic events
- Down syndrome (trisomy 21)
o The identification of a molecular or chromosomal abnormality is
- Edwards syndrome (trisomy 18)
a direct observation of the source of some diseases
- Patau syndrome (trisomy 13)
o The diploid human genome including twenty-three pairs of
- Klinefelter syndrome (XXY syndrome)
chromosomes and each is composed of 20 to 25 thousand genes
- Turner syndrome (monosomy X) and trisomy X
o An allele is positioned on a locus, the specific location of a gene
or DNA sequence on a chromosome; so the diploid genome
MUTATION DETECTION
contains two alleles of each gene
o CYTOGENETICS – Karyotyping
o Chromosomes 1 to 22 are called autosomes and the twenty-third
o MOLECULAR
pair is the sex chromosomes (X and Y)
- Polymerase chain reaction (PCR)
o If the DNA sequence is mutated and the alteration is not repaired
- DNA sequencing
by the cell, subsequent replications reproduce the mutation
- DNA Microarrays
o Mutations can range from a single nucleotide alteration,
- Denaturing gradient gel electrophoresis (DGGE)
duplication or rearrangement of chromosomes
- Single strand conformation polymorphisms (SCCP)
o Genetic diseases are usually categorized into single-gene,
- Restriction fragment length polymorphisms (RFLP)
chromosomal, and multi- factorial disorders (mix with
environmental factor)
CYTOGENETICS
o Chromosome studies are advised in the following situations:
MUTATION
suspected chromosome abnormality, sexual disorders, multiple
o Heritable changes in DNA nucleotide sequence
congenital anomalies and/or developmental retardation,
o Variation in the DNA sequence of one or more nucleotides
undiagnosed learning disabilities, infertility
- Deletion - Substitution
o The microscopic study of chromosomes is performed on compacted
- Insertion - Translocation
chromosomes at a magnification of about 1000 at metaphase
- Duplication
CONVENTIONAL KARYOTYPING
POLYMORPHISMS
o A karyotype is the number and appearance of chromosomes in
o Variation in the DNA sequence that occurs naturally at high
the nucleus of a eukaryotic cell.
frequencies
o The term is also used for the complete set of chromosomes in a
o Human Leukocyte Antigen is a highly polymorphic region of
species or in an individual organism and for a test that detects
human DNA
this complement or measures the number.
o SNP is a type of mutation but not all mutations are SNPs
o Karyotype is a test to identify and evaluate the size, shape, and
- SNPs – single nucleotide polymorphism
number of chromosomes in a sample of body cells.
- LINES – long interspersed nucleotide sequences
o Extra or missing chromosomes, or abnormal positions of
chromosome pieces, can cause problems with a person's growth,
SINGLE GENE DISORDERS
development, and body functions
o arising from errors in DNA sequence of a gene are categorized into:
o Preparation of a visual karyotype is done by arresting dividing
- autosomal dominant (AD)
cells at metaphase stage with a microtubule polymerization
- autosomal recessive (AR)
inhibitor such as colchicine; the cells, then, are spread on a glass
- X-linked recessive (XR)
slide and stained with Giemsa stain (G-banding)
o chromosomes are studied by making a photograph or digital
AUTOSOMAL DOMINANT
imaging and subsequent assembling of chromosomes. Human
o A child of an affected
chromosomes are categorized based on position of centromere;
individual and an
in metacentric chromosomes, centromere is located in the
unaffected mate has a
middle (chromosomes 1, 3, 16, 19 and 20), chromosomes 13, 14,
50%–100% recurrence
15, 21, 22 and Y are acrocentric (the centromere near one end),
risk or likelihood of
and other chromosomes are submetacentric.
expressing the disease
phenotype
NORMAL KARYOTYPE
AUTOSOMAL RECESSIVE
o is the largest category of
mendelian disorders. The
recurrence risk is 25% if
siblings are affected,
indicating the presence of
the recessive mutation in
at least one of the parents.

X-LINKED RECESSIVE
DOWN SYNDROME
o are X-linked because
relatively few genes are
carried on the Y
chromosome. X-linked
mutations are almost
always recessive
o Even though one X
chromosome is inactivated in females, the inactivation is
reversible so that a second copy of X-linked genes is available. In
contrast, males are hemizygous for X-linked genes, having only
one copy on the X chromosome. Males, therefore, are more
likely to manifest the disease phenotype

[SARANILLO, KA.]
 MOLECULAR STUDIES SINGLE-STRAND CONFORMATION POLYMORPHISMS (SSCP)
RESTRICTION FRAGMENT LENGTH POLYMORPHISM o Sensitive method of mutant detection without sequencing; ideal
o RFLP is a technique invented in 1984 by the English scientist Alec for shorter fragments with <300bp
Jeffreys during research into hereditary diseases o The method is based on the preference of DNA (as well as RNA)
o It is used for the analysis of unique patterns in DNA fragments in to exist in a double stranded, rather than single-stranded
order to genetically differentiate between organisms o In the absence of a complementary strand, nucleic acids form
o Genetic polymorphism is defined as the inherited genetic intra-strand duplexes to attain as much of a double-stranded
differences among individuals in over 1% of normal population condition as possible
o The RFLP technique exploits these differences in DNA sequences o Each folded strand forms a three-dimensional structure, or
to recognize and study variation conformer, the shape of which is determined by the primary
o Variations occurring within a species in the length of DNA sequence of the folded strand
fragments generated by a specific endonuclease o SSCP is determined by the migration of the single-stranded
o Types of RFLP: RFLP using Hybridization techniques conformers in polyacrylamide gels under precisely controlled
PCR-RFLP denaturing and temperature conditions
o The shape of the conformer depends on the complementary
PROCEDURE FOR RFLP USING HYBRIDIZATION TECHNIQUES nucleotides available for hydrogen bonding and folding
o The first step in this process is to isolate the DNA from the target. o A single bp difference in the DNA sequence can cause the
o Once the the DNA is isolated from the sample it is subjected to conformer to fold differently
restriction digestion using restriction enzymes. o These conformers are resolved in a polyacrylamide gel or by
o The digested DNA sample is then subjected to gel electrophoresis, in capillary electrophoresis with temperature control
which the DNA is separated based on its size. Many DNA fragments o Differences in the shape of the conformers (kinks, loops,
with slight differences in length are produced. bubbles, and tails) are caused by sequence differences in the
o The gel is then exposed to a chemical to denature double- DNA single strand
stranded DNA to become single- stranded o Band or peak patterns different from those of normal sequence
o This is followed by southern blotting where DNA is transferred control conformers prepared simultaneously with the test
from gel to nylon membrane. conformers indicate the presence of mutations.
o The nylon membrane is then exposed to solution with
radioactive complementary nucleotide probes that hybridize to PROCEDURES FOR SSCP
specifically chosen DNA sequences on nylon membrane. The o Genomic DNA is subjected
membrane is then placed against X- ray film, where hybridized to PCR to amplify the
radioactive probes cause exposure of X-ray film, producing an target DNA
autoradiogram. o Amplicons are denatured
o RFLP analysis is carried out to detect differences in pattern to and cooled under non-
confirm polymorphisms. denaturing condition
leading to the formation of
different conformations
o Products are separated via
gel electrophoresis to
identify the presence of
mutants dependent on
their electrophoretic
mobility of separated based on their shape

DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE)

o Exploits differences in denaturation between a normal and
mutated DNA molecule caused by even one nucleotide
difference in a sequence
o contribution of the attraction between successive bases on the same
DNA strand can affect denaturation of double-stranded DNA
o double-stranded DNA fragments 200–700 bp in length are
PCR-RFLP
o The region prepared by PCR amplification of test sequences or by restriction
surrounding the digestion. The fragments are separated on polyacrylamide gels
mutation is containing a gradient of concentrations of urea and formamide
amplified, and the o A 100% denaturant solution is 7 M urea and 40% formamide.
mutation is Gradients range 15%– 90% denaturant, usually with a 10%–20%
detected by cutting difference between the high denaturant concentration at the
the amplicon with bottom of the gel and the low denaturant concentration at the
the appropriate
top of the gel for a given analysis
restriction enzyme
o As the double-stranded DNA fragment moves through the gel,
the denaturing conditions increase, sequences reach their
denaturing point, and the complementary strands begin to
denature
o Even a one-nucleotide difference between two DNA molecules
results in the two molecules denaturing at different positions in
APPLICATIONS OF RFLP
the gel. The band of the mutated DNA shifts to a different
o DNA FINGERPRINTING
- Forensic scientists may use RFLP analysis to identify suspects position in the gel as compared with the normal DNA band
based on evidence samples collected at scenes of crimes
o PATERNITY TESTING
- RFLP is also used in the determination of paternity or for
tracing ancestry.
o Genetic diversity
- technique can be used in studying evolution and migration of
wildlife, studying breeding patterns in animal populations and
in the detection and diagnosis of certain diseases

[SARANILLO, KA.]
 SPECIMEN COLLECTION
MOLECULAR BIOLOGY & DIAGNOSTICS 311 o Preserve viability/nucleic acid integrity of target microorganisms
DETECTION AND IDENTIFICATION OF MICROORGANISMS
FINALS: WEEK 16 LEC
o Avoid contamination
o Appropriate time and site of collection (blood, urine, other)
OBJECTIVES (CHAPTER 12)
o Use proper equipment (coagulant, wood, or plastic swab shafts)
o Identify the advantages and disadvantages of using
o Commercial collection kits are available
molecular-based methods as compared to traditional culture-
o The Clinical and Laboratory Standards Institute (CLSI) has
based methods.
guidelines for proper specimen handling
o Explain the value of controls, in particular amplification
controls, in ensuring the reliability of PCR results
SAMPLE PREPARATION
o Compare and contrast the molecular methods that are used
o Consider the specimen type (stool, plasma, CSF)
to type bacterial strains in epidemiological investigation
o More rigorous lysis procedures are required to penetrate cell
walls
TARGET MICROORGANISMS FOR MOLECULAR-BASED TESTING
o Consider the number of organisms in the sample
o Those that are difficult or time-consuming to isolate
o Inactivate inhibitors (acidic polysaccharides in sputum or
- e.g., Mycobacteria
polymerase inhibitors in CSF)
o Hazardous organisms
o Inactivate RNases
- e.g., Histoplasma, Coccidiodes
o Those without reliable testing methods
PCR DETECTION OF MICROORGANISMS: QUALITY CONTROL
- e.g., HIV, HCV
o PCR and other amplification methods are extremely sensitive
o High volume tests
and very specific. For accurate test interpretation, use proper
- e.g., S. pyogenes, N. gonorrhoeae, C. trachomatis
controls:
o Positive control: positive template
APPLICATIONS OF MOLECULAR BASED TESTING IN CLIN. MICROBIO.
o Negative control: negative template
o Rapid or high-throughput identification of microorganisms
o Amplification control: omnipresent template unrelated to target
o Detection and analysis of resistance genes
o Reagent blank: no template present
o Genotyping
o Classification
PCR QUALITY CONTROL: INTERNAL CONTROLS
o Discovery of new microorganisms
o Homologous extrinsic
- controls for amplification
MOLECULAR METHODS OF DETECTION OF MICROORGANISMS:
o Heterologous extrinsic
1. PCR—traditional, real-time, and reverse transcriptase PCR
- controls for extraction and amplification
2. DNA sequencing.
o Heterologous intrinsic
3. Pulsed-field gel electrophoresis (PFGE)
- human gene control
4. Matrix-assisted laser desorption ionization (MALDI) spectrometry
5. Pulsed-Field Gel Electrophoresis
QUALITY CONTROL: FALSE POSITIVES
6. Restriction Fragment Length Polymorphism Analysis
o Contamination: check reagent blank
7. Amplified Length Polymorphism Assay
o Dead or dying organisms: retest 3-6 weeks after antimicrobial therapy
8. Interspersed Repetitive Elements o Detection of less than clinically significant levels
9. Internal Transcribed Spacer Elements
o Improper collection, specimen handling
10. Spa Typing o Extraction/amplification failure: check internal controls
11. Multilocus Sequence Typing o Technical difficulties with chemistry or instrumentation: check
method and calibrations
COMPARISON OF MOLECULAR EPIDEMIOLOGY METHODS
ANTIMICROBIAL AGENTS
o Inhibit growth (-static); e.g., bacteriostatic, fungistatic
o Kill organisms (-cidal); e.g., bacteriocidal, fungicidal, viricidal
o Antimicrobial agents are classified by:
1. static/-cidal
2. mode of action
3. chemical structure

SITES OF ACTION OF ANTIMICROBIAL AGENTS

CONVENTIONAL METHODS OF DETECTION

ADVANTAGES DISADVANTAGES
Assesses living (culturable) Risk of contamination
microbes
Able to recognize viable cells in High skill level is necessary for
a sample optimal results
Easy to quantitate cells in a Time and resource intensive
sample MECHANISMS FOR DEVELOPMENT OF RESISTANCE TO
High sensitivity with Relies on phenotypic ANTIMICROBIAL AGENTS
appropriate media biochemical characterization o Enzymatic inactivation of agent
Assesses DNA sequences Risk of contamination o Altered target
(target-specific with PCR) o Altered transport of agent in or out
Potential to recover majority of Relies on established sequence o Acquisition of genetic factors from other resistant organisms
microbes identities
Modest skill level required Relies on well-designed primer ADVANTAGES OF MOLECULAR DETECTION OF RESISTANCE TO
set ANTIMICROBIAL AGENTS
Relatively quick and straight- DNA recovery influenced by o Mutated genes are strong evidence of resistance
forward processing cell lysis method o Rapid detection without culturing
Can freeze samples for later Unable to distinguish live from o Direct comparison of multiple isolates in epidemiological
processing dead cells investigations
High specificity

[SARANILLO, KA.]
 MOLECULAR EPIDEMIOLOGY OTHER GENOTYPIC METHODS USED TO TYPE ORGANISMS
o EPIDEMIC – rapidly spreading outbreak of an infectious disease o Plasmid fingerprinting with restriction enzymes
o PANDEMIC – a disease that sweeps across wide geographical areas o RFLP analysis
o EPIDEMIOLOGY – collection and analysis of environmental, o Amplified Fragment Length Polymorphism (AFLP)
microbiological, and clinical data o Interspersed repetitive elements
o Ribotyping
MOLECULAR EPIDEMIOLOGY o Spa typing
o Phenotypic analysis measures biological characteristics of o Multilocus sequence typing
organisms
o Molecular epidemiology is a genotypic analysis targeting VIRUS
genomic or plasmid DNA o “Classical methods” of detection include antibody detection,
- Species, strain, or type-specific DNA sequences are the antigen detection, or culture
sources of genotype information o Molecular methods of detection include target, probe, and signal
amplification
PULSED-FIELD GEL ELECTROPHORESIS (PFGE) o Tests are designed for identification of viruses, determination of
viral load (number of viruses per ml of fluid), and genotyping by
sequence analysis

TEST PERFORMANCE FEATURES FOR VIRAL LOAD MEASUREMENT

CHARACTERISTIC DESCRIPTION
Sensitivity Lowest level detected at least 95% of the time
Accuracy Ability to determine true value
Precision Reproducibility of independently determined test
results
Specificity Negative samples are always negative and positive
CRITERIA FOR PFGE PATTERN INTERPRETATION: RULE OF THREE results are true positives
Linearity A serial dilution of standard curve closely
approximates a straight line
Flexibility Accuracy of measurement of virus regardless of
sequence variation

VIRAL GENOTYPING
o Viral genes mutate to overcome antiviral agents
o Gene mutations are detected by sequencing
o Primary resistance mutations affect drug sensitivity but may
slow viral growth
o Secondary resistance mutations compensate for the primary-
resistance growth defects

SUMMARY
o Molecular-based methods offer sensitive and direct detection of
ARBITRATILY PRIMED PCR: RANDOM AMPLIFICATION OF microorganisms
POLYMORPHIC DNA (RAPD) o Due to high sensitivity and specificity, proper quality control is
critical for molecular testing.
o Several molecular methods are used to type bacterial strains in
epidemiological investigations
o Target, probe, or signal amplification procedures are also used
to determine viral load.

INTERSPERSED REPETITIVE ELEMENTS

o REP sequence inverted repeat

o ERIC sequence inverted repeat

- PCR amplification priming outward from repetitive

elements generates strain-specific products.

o Is the unknown (U) strain A or B?

[SARANILLO, KA.]
 THE EGFR GENE FAMILY
MOLECULAR BIOLOGY & DIAGNOSTICS 311
MOLECULAR ONCOLOGY TESTING
FINALS: WEEK 17 LEC

OBJECTIVES
o Contrast tissue-specific and tumor-specific molecular targets
o List molecular targets that are useful for diagnosis and the
monitoring of solid tumors
o Explain how microsatellite instability is detected
o Show how clonality is detected using antibody and T-cell
receptor gene rearrangements
o Describe translocations associated with hematological
malignancies that can be used for molecular testing

CANCER IS CAUSED BY NONLETHAL GENETIC MUTATIONS

AFFECTING CERTAIN GENES
o Oncogenes, as proto-oncogenes, normally promote cell division
or cell survival
- Oncogene mutations are usually again of function and
dominant
o Tumor suppressors: genes normally arrest cell division
- Tumor suppressor gene mutations are usually a loss of MOLECULAR ABNORMALITIES IN SOLID TUMORS
function and recessive EGFR
o The EGFR oncogene encodes another of the same family of
CANCER IS CAUSED BY NONLETAL GENETIC MUTATIONS epidermal growth factor receptors
o This gene is mutated or amplified in several types of cancer cells
o Tumors with activating mutations in EGFR are sensitive to
tyrosine kinase inhibitors
o EGFR protein is detected by IHC
o EGFR gene and chromosome abnormalities are detected by FISH
o EGFR gene mutations are detected by SSCP, SSP-PCR, or direct
sequencing

K-ras
o The Kirsten rat sarcoma viral oncogene (K-ras) encodes a key
component of cell signaling.
o Mutations in K-ras are the most common oncogene mutations in
cancer
o K-ras mutations are associated with tumor malignancy and may
affect response to some therapies
o K-ras gene mutations are detected by SSCP or direct sequencing

TP53
MOLECULAR DETECTION OF DISEASE o The 53-kilodalton tumor suppressor gene (TP53) encodes a
o Targets: transcription factor
- Tissue-specific markers (antigens, gene rearrangements) o TP53 is mutated in half of all types of cancer
- Disease-specific markers (translocations, point mutations, o Loss of TP53 function is an indicator of poor prognosis in colon,
polymorphisms in tumor suppressor or oncogenes) lung, breast, and other cancers
- Viruses (EBV, HCV, HTLV-1) o Mutant p53 protein is detected by IHC
o Methods o TP53 gene mutations are detected by a variety of methods,
- Hybridization, blotting including SSCP and direct sequencing
- Standard PCR, RT-PCR, electrophoresis
- PCR with heteroduplex analysis, SSCP OTHER GENES ASSOCIATED WITH SOLID TUMORS
- Real-time PCR with gene or patient-specific probes o Ewing sarcoma, EWS
o Synovial sarcoma translocation, chromosome 18; synovial
GENE AND CHROMOSOME ABNORMALITIES OBSERVED IN CANCER sarcoma breakpoint 1 and 2, SYT-SSX1, SYT-SSX2
o Gene mutations (oncogenes, tumor suppressor genes) o Paired box-Forkhead in rhabdomyosarcoma, PAX3-FKHR, PAX7-
o Chromosome structural abnormalities (translocations, deletions, FKHR
insertions) o Ataxia telangiectasia mutated gene, ATM
o Chromosome number abnormalities (aneuploidy, polysomy) o V-myc avian myelocytomatosis viral-related oncogene,
neuroblastoma-derived, MYCN or n-myc
MOLECULAR ABNORMALITIES IN SOLID TUMORS, HER2/neu o Rearranged during transfection (RET) protooncogene
o The HER2/neu gene encodes one of a family of human epidermal
growth-factor receptors INHERITED CANCER GENE MUTATIONS
o This gene is frequently amplified in breast cancer cells, resulting o Inherited tumor suppressor gene mutations are recessive for the
in increased amounts of HER2 cell surface protein malignant phenotype
o HER2-expressing tumors are sensitive to herceptin, a o Tumor suppressor gene
monoclonal antibody therapy mutations are dominant with
o HER2 protein is detected by immunohistochemistry (IHC) respect to increased risk of
o HER2/neu gene amplification is detected by fluorescence in situ malignancy
hybridization (FISH) o Loss o heterozygosity exposes
the recessive mutant allele in a
hemizygous state
o This is explained by the two-
hit hypothesis →

[SARANILLO, KA.]
 LOSS OF HETEROZYGOSITY CAN BE DETECTED BY STR ANALYSIS

Analytical Targets of Molecular Testing

o Although not yet completely standardized,
o These include tests that target tissue-specific or tumor-specific
genes.
INHERITED BREAST CANCER RISK o Tissue-specific targets are molecular characteristics of the type
o BRCA1 and BRCA2 are tumor suppressor genes encoding of tissue from which a tumor arose.
proteins that participate in DNA repair o Tumor-specific genes are not expressed in normal cells and are
o Inherited mutations in BRCA1 and BRCA2 significantly increase more definitive with respect to the presence of tumor.
risk of breast cancer at an early age
o Frequently occurring mutations, including 187delAG and Gene and Chromosomal Mutations in Solid Tumors
5382inC in BRCA1 and 6174delT in BRCA2, are detected by SSP- o A number of tests are routinely performed to aid in the
PCR and other methods diagnosis, characterization, and monitoring of solid tumors.
o Most mutations are detected by direct sequencing of both genes o One of many of this tumors is Ewing Sarcoma, EWS (22q12). A
group of tumors arising from primitive neuroectodermal tissue
(PNET)/Ewing’s sarcomas comprise a family of childhood
neoplasms referred to as the Ewing family.
o Detection of specific translocations by cytogenetic ormolecular
methods is useful for diagnostic and prognostic accuracy (Table
14.2). Gene and Chromosomal Mutations in Solid Tumors

Slide 17

ONCOLOGY
o is the study of tumors.
o A tumor, or neoplasm, is a growth of tissue that exceeds and is
not coordinated with normal tissue.
o Benign (not recurrent) or malignant (cancer) (invasive and
tending to recur at multiple sites).
o Molecular oncology is the study of cancer at the molecular level,
using techniques that allow direct detection of genetic
alterations, down to single base pair changes.

CLASSIFICATION OF NEOPLASMS
Two broad groups
o Solid tumors - according to the tissue of origin
- Carcinomas (epithelial) or sarcomas (bone, cartilage,
Microsatellite Instability
muscle, blood vessels, fat)
o Lynch syndrome, or hereditary nonpolyposis colorectal cancer
- Teratocarcinomas consist of multiple cell types.
(HNPCC), is an inherited form of colon carcinoma, accounting for
o Hematological malignancies (leukemias and lymphomas)
about 5% of all colon cancers.
- Leukemia is a neoplastic disease. Large number of WBC
o Predisposition to cancer in this syndrome is caused by mutations
- Lymphoma is a neoplasm of lymphocytes that forms
in the MSH2 and MLH1 genes
discrete tissue masses.
o Metastasis is the movement of dislodged tumor cells from the
original (primary) site to other locations. Only malignant tumors
are metastatic.

Molecular Basis of Cancer

o Cancer is caused by nonlethal mutations in DNA.
o The mutations affect two types of genes: oncogenes and tumor
suppressor genes
o Oncogenes promote cell division.
o Oncogenes also support cell survival by inhibiting apoptosis, or
self- directed cell de
o Tumor suppressors slow down or stop cell division.

[SARANILLO, KA.]