Introduction to Molecular

Biology and Immunology

Aniruddha Roy
 Tools and
Techniques of
Molecular
Biology
 Tools of cell biology

Compound microscope

Inverted microscope
Light microscopic image after staining of tissue
 Bright field microscope do not
have sufficient contrast without
staining
Bright field

Phase contrast

Differential interference contrast

Most importantly, we can not
distinguish between different
cellular structures
Fluorescence microscope
Endothelial cells under the microscope. Nuclei are stained blue
with DAPI, microtubules are marked green by an antibody bound
to FITC and actin filaments are labeled red with phalloidin bound
to TRITC. Bovine pulmonary artery endothelial (BPAE) cells
Limitation of fluorescence microscopy

Biological samples are not naturally

fluorescence
 Protein of interest labeled with fluorescent-
antibody
 Cell fixing; Blocking; Ant-Ab interaction;
Microscopy
 Protein of interest tagged with GFP
 Genetic manipulation; Chimeric protein;
Dynamic imaging
Confocal microscope
Fluorescence resonance energy transfer (FRET)
 The precise location
and nature of the
interactions between
specific molecular
species in living cells :
Conventional
fluorescence
microscopy enables
localization of
fluorescently labeled
molecules within the
optical spatial
resolution,
approximately 200
nanometers (0.2
micrometer).

However, in order to understand the physical interactions between protein

partners involved in a typical biomolecular process, the relative proximity
of the molecules must be determined more precisely. The technique
of fluorescence resonance energy transfer (FRET), when applied to
optical microscopy, permits determination of the approach between two
molecules within several nanometers.
The different types of
fluorescent-protein based FRET-
biosensors. FRET biosensors
indicate a biological change
through a change in the FRET
signal caused by an alteration
of the donor-acceptor distance
by (A) cleavage of a linker
domain; (B) a conformational
change due to internal
alterations or
(C) a conformational change
due to the effect of an external
mechanical force.
(D) by a change of the
fluorescence properties
triggered by a change of the
surrounding environment of the
fluorophores.
Electron microscopy
Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM
Transmission Electron Microscopy
Its basically captures shadow
Staining with heavy metal salt : either negative or positive
Scanning Electron Microscope
Surface morphology; Finer details
Coating the sample with gold or platinum
Recombinant DNA Technology
Cloning Technology
Generation of many copies of DNA template (e.g., recombinant
DNA molecule) that is replicated in a host.

 Developed in the 1970s.

 Prior to discovery of PCR methods.
 Can generate unlimited supply of gene copies.
 Many applications:

• Genetic mapping
• DNA sequencing
• Mutation studies
• Transformation
• Genetic engineering
• Genome sequencing

 Prior to the development of this technology, DNA was

difficult to work with because it was hard to obtain sufficient
copy number to visualize or manipulate.
DNA Cloning
Goal is to generate large amounts of pure DNA that can be
manipulated and studied.

DNA is cloned by the following steps:

1. Isolate DNA from organism

2. Cut DNA with restriction enzymes to a desired size.
3. Ligate each piece of DNA into a cloning vector to create a
recombinant DNA molecule.
Cloning vector = artificial DNA molecule capable of
replicating in a host organism (e.g., bacteria).
4. Transform recombinant DNA (cloning vector + DNA
fragment) into a host (e.g., bacteria) that will replicate and
make copies.
5. E. coli is the most common host.
Step 1-Isolate whole genomic DNA from organism

DNA extraction easily performed using:

• SDS (detergent) to break up cell membrane and

organelles and to precipitate proteins.

• Salt (NaCl) lyses cells and binds the DNA strands

together.

• Proteinase K to digest proteins bound to DNA (essential to

remove eukaryotic chromatin).

• Ethanol (EtOH) to precipitate and wash DNA.

• Water to resuspend and store DNA.

Step 2 - Cut DNA with restriction enzymes
Cut and ligate 2 different DNAs with EcoRI ---> recombinant DNA
Step 3-Ligate DNA into some kind of cloning vector to create
a recombinant DNA molecule

Six different types of cloning vectors:

1. Plasmid cloning vector

2. Phage cloning vector

3. Cosmid cloning vector

4. Shuttle vectors

5. Yeast artificial chromosome (YAC)

6. Bacterial artificial chromosome (BAC)

7. Fosmid cloning vector

Phage cloning vector
Cosmid cloning vector
Shuttle vectors
Plasmid Cloning Vectors:

 Bacterial plasmids, naturally occurring small ‘satellite’

chromosome, circular double-stranded extra-chromosomal
DNA elements capable of replicating autonomously.
 Plasmid vectors engineered from bacterial plasmids for use
in cloning.
 Features (e.g., E. coli plasmid vectors):
1. Origin sequence (ori) required for replication.
2. Selectable trait that enables E. coli that carry the plasmid
to be separated from E. coli that do not (e.g., antibiotic
resistance, grow cells on antibiotic; only those cells with
the anti-biotic resistance grow in colony).
3. Unique restriction site such that an enzyme cuts the
plasmid DNA in only one place. A fragment of DNA cut
with the same enzyme can then be inserted into the
plasmid restriction site.
4. Simple marker that allows you to distinguish plasmids
that contain inserts from those that do not (e.g., lacZ+
Cloning
in
plasmid
vectors
 Polylinker:
restriction sites

lacZ+
gene

Origin sequence
Ampicillin
resistance gene
 *Cut with same
restriction enzyme
*DNA ligase

An intensely blue
X-gal product which is
Incorporation of the plasmid in E Coli by
electroporation:
What do you create by cloning?

Recombinant DNA Libraries (2 major types):

1. Genomic/chromosomal library, Collection of cloned

restriction enzyme digested DNAs containing at least one
copy of every DNA sequence in a genome or chromosome.
2. Complementary DNA (cDNA) library, Collection of
clones of DNA copies made from mRNA isolated from
cells.
 reverse transcriptase (RNA dependent DNA
polymerase)
 Synthesizes DNA from an RNA template
 
Number
cDNAof clones reflect
libraries required for is
what a complete library can
being expressed be
in cells.
calculated from the size of the genome and average size
of overlapping fragments cut by restriction enzymes.

 Library should contain many times more clones than the

calculated minimum number of clones.
Screening a genomic library :
1. Plasmid vectors containing digested genomic DNA are
transformed into E. coli and plated on selective medium
(e.g., ampicillin).
2. Colonies that grow are then are transferred and replicated
onto a nylon membrane (E. coli continues to grow on the
membrane).
3. Bacteria are lysed and DNA is denatured.
4. Nylon membrane bound DNA is next probed with
complementary DNA (e.g., 32P radio-labeled DNA or
flourescent probe).
5. Complementary DNA in the probe is composed of DNA
sequence you are looking for.
6. Unbound probe DNA is washed off the filter.
7. Hybridization of probed DNA is detected by exposure to X-ray
film or by chemiluminescence.
8. Pattern is noted from exposure pattern of clones on X-ray
Screening a
genomic
library
3. Transfer clones
to nylon membrane

4. Denature DNA
and fix in place on
nylon membrane in
hybridization oven.

5. Hybridize labeled
probe to DNA.

6. Identify position
of labeled probe with
X-ray firm or
chemical
illuminescence.
7. Locate position of
corresponding clone
in original bacterial
colony.
cDNA Library:
1. cDNA is derived from mature mRNA, does not include
introns.
2. cDNA may contain less information than the coding region.
3. cDNA library reflects gene activity of a cell at the time
mRNAs are isolated (varies from tissue to tissue and with
time).
4. mRNA degrades quickly after cell death, and typically
requires immediate isolation (cryoprotectants can increase
yield if immediate freezing is postponed by fieldwork).
5. Creating a cDNA library:
1. Isolate mRNA
2. Synthesize cDNA
3. Clone cDNA
Creating a cDNA library - Step 1-Isolate
mRNA:
Creating
a cDNA
library -
Step 2-
cDNA
synthesis
:
PCR
RT-PCR
RT-PCR cont.
cDNA
RT-PCR

ethidium bromide
Mid-Sem syllabus
 Nucleic Acid Detection
• Hybridization of a labeled nucleic acid to
complementary sequences can identify specific
nucleic acids.
• probe – A radioactive nucleic acid, DNA or RNA,
used to identify a complementary fragment.

Southern Blot
• Used to detect a specific DNA sequence in a
sample.
• Use restriction endonuclease to cut DNA into
fragments.
• DNA fragments separated by electrophoresis.
Electrophoresis through a Gel Separates DNA
and RNA Molecules According to Size
• Gel electrophoresis
separates DNA
molecules according to
their size (including
molecular weight,
shape, charge,
topological properties
etc.)
subjected to an electric
field through a gel
matrix
• reveal the bands by
staining the gel with
fluorescent dyes, such
as ethidium
The gel is then over­laid with a
nitrocellulose filter or nylon
membrane to which the DNA
fragments are transferred
(blotted) to yield a replica of the
gel.
The filter is then incubated with
a labeled probe, which
hybridizes to the DNA fragments
that contain the complementary
sequence, allowing visualization
of these specific fragments of
cell DNA.
 In-Situ Hybridization
• Target nucleic acid found in intact cells.
• Provides information about presence of
specific DNA targets and distribution in
tissues.
• Probes must be small enough to reach
nucleic acid.
• Radioactive or fluorescent tags used.
• Requires experience.
 In-Situ Hybridization

• Fluorescent in-situ hybridization (FISH)

very popular.
• Cytogenetic technique used to detect and
localize presence or absence of DNA
sequences on chromosomes.
• Used in genetic counseling, medicine and
species identification.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC395705/pdf/520243.pdf
Microarray Differentia
l
expression
of genes
Same stapes with
control cells.
cDNA labeled with
different dye
Subtractiv Differentia
e l
hybridizati expression
on of genes

mRNA
DNA sequencing
Automated DNA sequencing
Column chromatography
A porous column of
beads equilibrated
in a particular
solvent is prepared,
and a sample
containing a
mixture of proteins
is applied to the
top of the column.
The sample is then
washed through
the column, and
the column eluate
is collected in a
succession of test
tubes. Because of
the properties of
the beads in the
column, proteins
with different
properties elute at
varying rates off
the column.
Three types of beads used for column chromatography. (A) The beads may
have a positive or a negative charge. Proteins that are positively charged in
a pH 7 buffer will flow through the column; negatively charged proteins will
be bound to the beads and can be subsequently eluted with a gradient of
salt. (B) The beads can have cavities or channels of a defined size; proteins
larger than these channels will be excluded from the beads and elute in the
“void volume” of the column; smaller proteins of various sizes will, to
varying degrees, enter the beads and pass through them, thereby
becoming delayed in their elution from the column. Such columns,
therefore, resolve proteins by size. (C) The beads can be derivatized with a
molecule that specifically binds the protein of interest. In the example
shown, it is a substrate (or substrate analog) for a particular enzyme; the
beads could also be derivatized with an antibody to the protein of interest,
in which case this would be called immunoaffinity chromatography.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)
Western blotting. (1) Proteins are resolved by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). The gel with the resolved set of proteins is then placed in an
apparatus that permits electrophoretic transfer of the proteins from the gel to the surface of
a special paper (e.g., nitrocellulose paper) to which proteins strongly adsorb. (2) After transfer
the nitrocellulose sheet is incubated with an antibody (the “primary” antibody) directed
against the protein of interest. (Before this incubation [not shown], the surface of
nitrocellulose paper is “blocked” by incubating it with a nonreactive protein such as casein, to
prevent nonspecific binding of the 1° antibody to the nitrocellulose; this casein block leaves
the sample proteins still available for antibody binding.) (3) After washing away unbound 1°
antibody, an enzyme-linked 2° antibody is added, which binds the 1° antibody and (4) can
generate a colored product for detection.
Fluorescence-activated cell sorting (FACS)
Transgeni
c animal
Knock down of gene
Antisense technology
siRNA technology

RISC: RNA-induced silencing comple

When double-stranded RNAs are introduced into cells, they are
cleaved into short double-stranded molecules (21-23
nucleotides) by an enzyme called Dicer. These short double-
stranded molecules, called short interfering RNAs (siRNAs),
then associate with a complex of proteins known as the RNA-
induced silencing complex (RISC). Within this complex, the two
strands of siRNA separate and the strand complementary to
the mRNA (the antisense strand) guides the complex to the
target mRNA by complementary base pairing. The mRNA is
then cleaved by one of the RISC proteins. The RISC-siRNA
complex is released following degradation of the mRNA and
can continue to participate in multiple rounds of mRNA
cleavage, leading to effective destruction of the targeted
Knock out of gene
Check this out!!!

https://www.labxchange.org/library/clusters/lx-
cluster:abe