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Biomol 12 - Molecular Biology Techniques

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29 views76 pages

Biomol 12 - Molecular Biology Techniques

Uploaded by

Erin Armaida
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Molecular

Biology
Techniques
Lecture 12
Molecular Biology

Department of Biology
Faculty of Mathematics and Natural Sciences
Universitas Indonesia
Contents

Nucleic
PCR acid
Labelling
DNA
Sequencing
Blotting
(Western,
and
Southern)
DNA Sequencing
• Determining sequence of bases in DNA that enables the
processive identification of each base in turn.

• Three main requirements to achieve processive


identification:
• DNA fragmens need to be prepared in a form suitable for
sequencing
• The technique used must achieve the aim of presenting each base
in turn in a form suitable for identification
• The detection method must permit rapid and accurate identification
of the bases.
DNA Sequencing: principles

Sequencing technique will Detection step is the final stage


generate nested fragments, which of sequencing procedure,
is overlapping fragments that usually involves separation of
terminate at different bases and fragments on a polyacrylamide
differ in length by one nucleotide. gel.

Two main methods for DNA sequencing:


enzymatic and chemical method.
Preparation of DNA fragments
Two strategies for sequencing large stretches of DNA
Ordered Sequencing Strategy Shotgun Sequencing Method

Fragments are tracked


and their relative order
is noted as the project
progress, and then put
together by reference to
the order of the
fragments.

Fragments are generated


and processed at random,
assembly of the sequence
is then carried out by
searching for sequence
overlaps using a
computer.
DNA Sequencing
Maxam-Gilbert Sanger-Coulson
(chemical) (enzymatic)
sequencing sequencing
Maxam-Gilbert (chemical) sequencing

• Starting material: defined fragment of DNA.


• Applicable to any DNA fragment (doesn’t
need to be cloned in a plasmid vector).
• Theory: given the large number of
molecules and different reactions, this
process will produce a set of nested
fragments.
a. DNA is radiolabelled with 32P at 5’
ends of each strand and then the
strands are denatured, separated,
and purified to give a population of
labelled strands for the sequencing
reactions.
b. Chemical modification of bases in
DNA strand in series of four to five
reactions with different specificities,
with on average, one base being
affected per molecule.
c. Modified bases are removed from
their phosphodiester backbone and
the strands cleaved at these
positions using piperidine.
d. The process produces a set of
fragments differing in length by one
nucleotide, labelled at their 5’
termini.
Maxam-
Gilbert
(chemical)
sequencing *ATTGACTTAGCC*ATTGACTTAGCC
*ATTGACTTA *ATTGACTTA
*ATTGACTTAGCC*ATTGACTTAGCC
*ATTGACTTAGC *ATTGACTTAGC
*ATT *ATTGACTT *ATTGACTTAG *ATTGACTTAG
*ATTG *ATTGACT *ATTGA
*ATT *ATTGAC
*ATTGA
*AT
*A
Sanger-Coulson (enzymatic) sequencing
• Similar END RESULT, totally different PROCEDURE.

• More complicated and usually involves subcloning into

different vectors like plasmid.


• Another method is to clone the DNA into vector such as

bacteriophage M13 which produces ssDNA during


infection→provides suitable substrate for the sequencing

reactions.
Sanger-Coulson (enzymatic) sequencing
a. A Primer is annealed to a ssDNA
template.
b. The Klenow fragment of DNA
polymerase I is used to synthesize a
copy of the DNA. A radiolabelled
dNTP (usu. [α-35S]dNTP, solid
circles) is incorporated into the DNA.
c. Chain termination occurs when
dideoxynucleoside triphosphate
(ddNTP) is incorporated.
d. A series of four reactions, each
containing one ddNTP in addition to
the four dNTPs required for chain
elongation, generates a set of
radiolabelled fragments.
DNA synthesis requires a 3´-OH to make the next
phosphodiester bond during DNA synthesis

normal
dNTP
H

ddNTPs block formation of the next phosphodiester bond


during DNA synthesis.

A 3´-OH on the last ribose is needed for DNA synthesis.


A mixture of dNTPs and ddNTPs
are used in DNA sequencing
Sanger-Coulson (enzymatic) sequencing

Reading DNA Sequence


a. An autoradiograph of part of a
sequencing gel.
b. A tracing of the autoradiograph.
• Each lane corresponds to a
reaction containing one of the
four ddNTPs used in the chain-
termination technique for DNA
sequencing.
• Read from the bottom of gel.
Electrophoresis and reading of sequences
• Separation → achieved by PAGE.
• Gels are 0.5 mm thick, usually contain 6-20% polyacrylamide and 7 M
urea, which acts as a denaturant to reduce the effects of DNA 2’
structure → important, because fragments that differ in length by only
1 base are separated.

1. Gel is run at hi-power settings → heat up 60-7-oC → maintain


denaturing condition.
2. After running gel, it is removed and may be dried onto a paper
sheet and then exposed to X-ray film.
3. Emissions from the radioactive label sensitize the silver grains,
which turn black when the film is developed and fixed →
autoradiograph (see prev. slide).
Polyacrylamide gel electrophoresis is used to visualize the
results of the sequencing reaction
Automation of DNA sequencing
• Enables large-scale production.
• Improvements in sample preparation and handling, with
robotic processing enabling hi-volume throughput.
• Linear continuous capillary electrophoresis techniques.
• Without the ability to analyse sequence data, the
sequence remains essentially silent/uninformative → dealt
with BIOINFORMATICS!
Automated DNA sequencing with fluorescent
dyes coupled to each reaction

Fluorescent dye coupled to


reaction allows visualization
of di-deoxy termination
events by means of a laser
that detects the colored
product.

This shows four different


reactions as done with the
old manual sequencing.
Automated DNA sequencing output
4 reactions carried out in one tube
The genomes
of many
organisms
have been
sequenced
Contents

Nucleic
PCR acid
Labelling
DNA
Sequencing
Blotting
(Western,
and
Southern)
PCR : A Molecular Xerox Machine for DNA

Some techniques to
analyze DNA and RNA
are limited by the small
amounts of test nucleic
acid available.

Polymerase chain Can replicate a


reaction (PCR) target DNA from a
rapidly increases few copies to
the amount of DNA millions in a few
in a sample. hours.
What is PCR?
▪ It is a molecular technology aim to
amplify a single or few copies of the
DNA to thousands or millions of copies.

▪ Developed in 1983 by Kary Mullis, PCR


is now a common and important
technique used in biological research
labs for a variety of applications.

▪ In 1993, Mullis was awarded the Nobel


prize in Chemistry along with Michael
Smith for his work on PCR.
NEW AUTOMATED PCR OLD PCR

Waterbath with 3 different


temperatures
Three Basic Steps that Cycle

Denaturation Priming Extension


• Heat to 98oC to • Primers added in a • 72oC
separate into two concentration that • DNA polymerase
strands. favors binding to extend the
• Cool to between the complementary molecule.
50oC and 65oC. strand of test DNA.
• Prepares the two
strands for
synthesis.

The amplified DNA can then be analyzed.


PCR
PCR increases the yield of DNA exponentially
PCR requirement
Component Function
DNA template • Any source of DNA that provides one or more target
molecules can in principle be used as a template for
PCR.
• Purity and quantity is important.
Primers • Specific to the section of DNA to be copied.
• Forward and reverse.
• Good primers are important.
Polymerase • Enzyme that reads original DNA sequence and makes
a complementary copy.
• DNA pol. from Thermus aquaticus is stable at 95oC
(Taq)
Pyrococcus furiosus (Pfu pol.), Thermus
thermophilus (Tth pol.), Thermus flavus (Tfl pol.)
dNTPs Nucleotides used to create the complementary strand of
DNA.
PCR buffer, MgCl2, Enables the reaction to take place.
Water
Designing PCR primers
1. Primers should be 17-28 bases in length;
2. Base composition should be 50-60% (G+C);
3. Primers should end (3') in a G or C, or CG or GC: this
prevents "breathing" of ends and increases efficiency of
priming;
4. 3'-ends of primers should not be complementary (ie. base
pair), as otherwise primer dimers will be synthesized
preferentially to any other product;
5. Primer self-complementarity (ability to form 2o structures
such as hairpins) should be avoided;
6. Tms between 55-80oC are preferred.
Designing PCR primers
1. Primers should be 17-28 bases in length;
Increase uniqueness.
2. Base composition should be 50-60% (G+C);
Also affecting:
- Melting temperature
- Stability

3. primers should end (3') in a G or C, or CG or GC (GC


clamp): this prevents "breathing" of ends and increases
efficiency of priming;
The presence of G or C bases within the last five bases from
the 3' end of primers (GC clamp) helps promote specific
binding at the 3' end due to the stronger bonding of G and C
bases.
Designing PCR primers
4. 3'-ends of primers should not be complementary (ie. base
pair), as otherwise primer dimers will be synthesized
preferentially to any other product;
5. Primer self-complementarity (ability to form 2 o structures
such as hairpins) should be avoided;
Self-Dimer Hairpin loop

ΔG Positive: not stable


ΔG Negative: stable
Designing PCR primers
6. Tms between 55-80oC are preferred;
The temperature at which one half of the DNA duplex will
dissociate to become single stranded and indicates the duplex
stability
Primers with Tm 52 – 58 oC generally produce the best results.
The GC content of the sequence gives a fair indication of the primer Tm.

The difference between Tm forward & Tm reverse should not bigger than 3oC.

Basic Tm calculation

Tm= (wA+xT)*2 + (yG+zC)*4


(Marmur J and Doty P (1962) J Mol Biol 5:109-118)

Catatan:w,x,y,z adalah jumlah basa


Annealing temperature (Ta)
Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 14.9
Tm product can be calculated if the sequence of product is known

where
Tm of primer is the melting temperature of the less stable
primer-template pair
Tm of product is the melting temperature of the PCR product
Rychlik W., Spencer W.J., Rhoads R.E., Spencer W.J., Rhoads R.E., Rhoads R.E. Optimization of the annealing temperature for
DNA amplification in vitro. Nucleic Acids Res. 1990;18:6409–6412.

Generally :
Tanneal = Tm_primer – 5C
Hot start PCR
A modified form of PCR which avoids a non-specific amplification
of DNA by inactivating the taq pol. at lower temperature.

The enzyme is not active in low temp. by adding specific


antibodies that block Taq-polymerase at annealing temperature.
But when the temperature raises for amplification to 72oC, the
specific antibody detaches from Taq-polymerase and the
amplification with greater specificity starts.

Hot Start PCR significantly reduces


nonspecific priming.
Real Time (reverse transcriptase) PCR

• RT-RT-PCR or just RT-PCR. Tissue / cells

• To quantitate differences in mRNA


extract RNA
expression → to have an idea about
gene expression (need to be
compared to the expression of copy into cDNA
reference genes, eg. housekeeping (reverse transciptase)
genes).
do real-time PCR

• Limited amount of mRNA


• Small amount of tissue/cells analyze results
• Precious reagents.
Real Time (reverse transriptase) PCR
To quantitate differences in mRNA expression ?

• PCR / reverse transcriptase PCR and subsequent

analysis by agarose gels → more qualitative

• Northern → agarose, blot → qualitative

• In situ hybridization → qualitative

• Real Time PCR gives quantitative results


Example (1) : the expression of genes encoding AIR12, Putative early
nodulin-like 2, Rhicadhesin receptor & Blue copper domain proteins in
2 different samples (2HA & SKL plants)
Example (2) : the expression of genes encoding Serine
carboxypeptidase in 4 organs (leaf, cotyledon, root & root tip) in
inoculated & uninoculated plants.

Serine carboxypeptidase

16

14
Relative expression

12

10

0
Leaf Ctrl Leaf Inoc Cotyl Ctrl Cotyl Inoc Root Root Root Tip Ctrl Root Tip
Segment Segment Inoc
Ctrl Inoc
Real Time PCR vs PCR

Web tools to design RT-PCR primers


https://www.genscript.com/ssl-
bin/app/primer
http://sg.idtdna.com/scitools/Applicati
ons/RealTimePCR/
http://www.quantprime.de/

Methods:
• Using fluorescent intercalating agent (eg. SYBR
green fluorescence)
• Using Taqman probe
RT-PCR using SYBR Green

SYBR green is a fluorogenic minor groove binding dye that exhibits


little fluorescence when in solution but emits a fluorescent signal upon
binding to double-stranded DNA (fluorescent intercalating agent).
RT-PCR using Taqman probe

The light emitted from the dye in


the excited state is received by a
Before the probe is met with the Taq polymerase, computer and shown on a graph
energy is transferred from a short-wavelength display, such as this, showing
fluorophore (green) to a long-wavelength fluorophore PCR cycles on the X-axis and a
(red). When the polymerase adds nucleotides to the logarithmic indication of intensity
template strand, it releases the short-wavelength on the Y-axis.
fluorophore, making it detectable and the long-
wavelength undetectable.
Taqman probes
• The PCR is prepared as usual, and the reporter probe is
added.
• As the reaction commences, during the annealing stage of the
PCR both probe and primers anneal to the DNA target.
• Polymerization of a new DNA strand is initiated from the
primers, and once the polymerase reaches the probe, its 5'-3'-
exonuclease degrades the probe, physically separating the
fluorescent reporter from the quencher, resulting in an increase
in fluorescence.
• Fluorescence is detected and measured in a real-time PCR
machine, and its geometric increase corresponding to
exponential increase of the product is used to determine the
quantification cycle in each reaction.
Nested PCR
PCR site directed mutagenesis
Common PCR
Restriction fragment length polymorphisms
(RFLPs)
Southern Blot
Consider two alleles of a gene.
Allele A has 3 BamHI sites, while
allele a has only two BamHI sites.

probe

HpaI Digest
Nor- Variants
mal 1 2 3
70% of carriers of the sickle cell gene
have a 13.0 kb HpaI fragment.
30% of carriers have 7.0 kb HpaI
fragment.
PCR applications
• Marker development: RAPD PCR, RFLP PCR, AFLP PCR, etc.
• DNA fingerprinting using molecular marker.
• Site directed mutagenesis → using primer that contains the mutations.

• Medical applications:

• For prenatal diagnosis, PCR used to amplify DNA from fetal cells
obtained from amniotic fluid.
• Tay-Sachs disease, phenylketonurea, cystic fibrosis, hemophilia,
Huntingdon's disease, Duchenne muscular dystrophy (DMD).
• the direct detection of HIV genomes in patient blood before the
appearance of HIV antibodies
• Forensic application
Variable Number of Tandem Repeat (VNTR)
analysis is commonly used in forensics

VNTR is based on hypervariable microsatellite sequence polymorphisms


within the human genome. These sequences (e.g., CACACA …) are found in
many locations in the human genome and vary greatly from person to person.
Using VNTR to compare forensic and
suspect samples

Individuals A & C are


excluded by this
analysis. The samples
from individual B will be
subjected to further tests.
PCR Results Detection
Contents

Nucleic
PCR acid
Labelling
DNA
Sequencing
Blotting
(Western,
and
Southern)
Labelling the Nucleic Acid
1)Keeping track (tracing) of small amounts of nucleic acid → low specific
activity is suffice

2) To produce highly radioactive nuc. acid molecules for hybridization


(radioactive probes) → high specific activity is necessary

Types of label:
• Radioactive (radiolabeling)
tritium (3H), carbon-14 (14C) → low energy emitter
sulphur-35 (35S) → medium energy emitter
phosphorus-32 (32P) → high energy emitter → more hazardous
• Non radioactive
Fluorescent dyes
Enzyme-linked labels → Elisa

Radioactive molecules → dNTP labelled with 3H or 32P


Labelling the Nucleic Acid – End Labelling

If the ATP donor is radioactively labelled, this produces a labelled nucleic


acid of relatively low specific activity, as only the termini of each molecule
become radioactive.
Labelling the Nucleic Acid – Nick translation

If labelling DNA by nick translation. (a) A single-strand nick is introduced into the
phosphodiester backbone of a DNA fragment using DNase I (low conc). (b) DNA
polymerase I then synthesizes a copy of the template strand, degrading the non-
template strand with its 5→3 exonuclease activity. If [α-32P]dNTP is supplied this
will be incorporated into the newly synthesized strand (solid circles).
Labelling the Nucleic Acid – Primer extension

Labelling DNA by primer extension (oligolabelling). (a) DNA is denatured to give


single-stranded molecules. (b) An oligonucleotide primer is then added to give a
short double-stranded region with a free 3-OH group. (c) The Klenow fragment of
DNA polymerase I can then synthesise a copy of the template strand from the
primer, incorporating [α-32P]dNTP (solid circles) to produce a labelled molecule with
a very high specific activity.
Nucleic Acid Hybridization
Contents

Nucleic
PCR acid
Labelling
DNA
Sequencing
Blotting
(Western,
and
Southern)
Blotting
Southern Blot
Southern Blot - Method
1. Restriction
2. Gel electrophoresis
3. DNA denaturation (alkaline
method, NaOH) – denature
dsDNA
4. Blotting to nitrocellulose
membrane
5. Fixation (using vacuum or
oven 80oC or UV radiation)
to permanently attach DNA
to membrane
6. Hybridization
7. Wash excess probe
Western
Blot
Western Blot – Detection, Why not radiolabeling?
• Membrane-bound proteins are generally detected using
secondary antibodies that are labeled with radioisotopes
or colloidal gold, or that are conjugated to fluorescent
molecules (fluorophores) or an enzyme such as alkaline
phosphatase (AP) or horseradish peroxidase (HRP).
• Early blotting systems used 125I-labeled reagents similar
to those used in radioimmunoassays.
• These systems provide sensitive results, but the special
handling and disposal problems of 125I reagents have
discouraged continued use of this technique.
Western
Blot
Mechanism of detection chemistries. In each method of western blot detection, a detectable
signal is generated following binding of an antibody specific for the protein of interest. In
colorimetric detection (A), the signal is a colored precipitate. In chemiluminescence (B), the
reaction itself emits light. In fluorescence detection (C), the antibody is labeled with a
fluorophore.
Gel Electrophoresis

• Nondenaturing GE / Native
• Agarose GE • Denaturing gradient GE
• Polyacrylamide GE • Pulse field GE
Denaturing gradient GE / DGGE
using chemical gradient
DNA is subjected to increasingly extreme denaturing
conditions, the melted strands fragment completely into single
strands.

• Able to recognise the differences in DNA sequences or mutations of various


genes.
• sequence differences in fragments of the same length often cause them to
partially melt at different positions in the gradient and therefore "stop" at
different positions in the gel.
• By comparing the melting behavior of the polymorphic DNA fragments
side-by side on denaturing gradient gels, it is possible to detect fragments
that have mutations in the first melting domain (Helms,1990).
• Placing two samples side-by-side on the gel and allowing them to denature
together, researchers can easily see even the smallest differences in two
samples or fragments of DNA.
Pulse Field GE
• used for the separation of large DNA molecules
• by changing periodically the electric field from a gel matrix.
• In a standard gel, DNA molecules bigger than 15 Kb move
together regardless of their size.
• PFGE is a variation that introduces alternating voltage
gradient to improve the resolution of larger molecules.
• The voltage is periodically switched among three directions:
(1)To the central axis of the gel, (2,3) at an angle of 60
degrees from both sides. The result is that DNA does not
move in a straight line through the gel but in an "net forward"
migration pattern.
Pulse Field GE
END OF THIS TOPIC
References
• Schleif R. Genetics and Molecular Biology, Second
Edition. The Johns Hopkins University Press.

• Weaver, R.F. 2001. Molecular Biology. McGraw-Hill.

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