GMB ASSIGNMENT-1

ELISA
ELISA is an abbreviation for "enzyme-linked immunosorbent assay."

An ELISA test uses components of the immune system (such as IgG or IgM antibodies) and
chemicals for the detection of immune responses in the body (for example, to infectious microbes).
The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves
an antibody or antigen (immunologic molecules) that may form an antigen-antibody reaction to
provide a positive result or, if they do not react, a negative result. Examples of the uses of an ELISA
test include diagnosing infections such as HIV and some allergic diseases like food allergies and
experimental investigations to identify compounds. ELISA tests are also known as an immunosorbent
assay or an enzyme immunoassay when an enzyme is bound to another substance as an indicator.

The test is based on a microtiter plate that has a solid phase substrate (target protein, antigen) at a
known concentration fixed to the plate that when exposed to an antibody that has an indicator
attached (dye for color change or enzyme-labeled antibody) that can produce a color change.
Depending on a standard curve for absorption of enzyme-labeled antibody versus antigen level as
related to the dye color change, tests may provide semi-quotative, quantitative, and/or identification of
many diverse substances. This type of test is termed a direct ELISA.

There are other types of ELISA tests. Indirect ELISA uses a secondary antibody to attach to the
substrate, and the sandwich ELISA that uses the antibody as the substrate fixed to the microtiter
plate.

ELISA tests primarily detect proteins (as opposed to small molecules and ions such as glucose and
potassium). The substances detected by ELISA tests can include hormones, an allergen, viral
antigens (dengue fever, for example), bacterial antigens (TB, for example), and antibodies that the
body has made in response to infection (antibodies to hepatitis B, for example) or vaccination. They
can also identify an infectious disease agent in patients.

Northern,Southern,Western Blotting
Southern Blotting – Southern blotting is a technique to detect if a particular sequence of DNA is
present (or a particular gene is present) in a sample of DNA. The various steps involved in this
process are similar to those employed in other blotting techniques.

Steps –

1) Breaking up DNA into pieces – The DNA is broken into small fragment by using restriction
enzymes. These enzymes cut DNA at specific sites.

2) Amplification by PCR – The DNA fragments are then copied in multitudes using polymerase
chain reaction.

3) Gel Electrophoresis – The DNA fragments are then segregated by using gel electrophoresis
in which the property that DNA is negatively charged is exploited and they get separated on the basis
of molecular weights when introduced to an electric field.

4) Denaturation – The gel is then soaked in an alkali or acid which denatures the double
fragmented DNA (strands get separated).

5) Blotting – The separated strands are then transferred to a positively charged nylon
membrane.

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6) Hybridization with labelled probe – The probes are basically complementary sequences of
gene of interest, they typically have a radioactive atom or fluorescent dye, this probe binds to the
complementary DNA (the gene) on the membrane.

7) Visualization – These DNA fragments can then be visualized under autoradiogram which
gives pattern of bands.

Northern Blotting – It is a method used to detect a specific RNA sequence among a mixture of RNA.
Northern blotting can be used to analyse a sample of RNA from a particular tissue in order to
measure the RNA expression of particular genes. The steps involved in northern blotting are very
similar to southern blotting. A few differences are that in the first step the RNA is separated into single
strands and then get fragmented and run through gel electrophoresis. The probe that is used is the
complementary RNA sequence. It is basically southern blot except it is being used to study gene
expression via mRNA transcripts.

Western blotting – Western blotting is a technique to detect and analyse proteins. The various steps
are as follows. Samples are prepared and loaded on to a gel. Then by the gel electrophoresis process
negatively charged proteins move towards the anode. Then they are transferred onto a membrane,
this is called blotting step.

Antibody probing – We form primary antibodies specific to that protein, these antibodies are then
transferred onto the polymer sheet which bind to the antigen that is specific to the protein. We then
form secondary antibody which are labelled and bind to the primary antibody. The complex is now
used for detection. The band to which our complex bonded was our protein to be detected.

Gel electrophoresis
Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such
as RNA and proteins) based on their size and charge. Electrophoresis involves running a current
through a gel containing the molecules of interest. Based on their size and charge, the molecules will
travel through the gel in different directions or at different speeds, allowing them to be separated from
one another.

All DNA molecules have the same amount of charge per mass. Because of this, gel electrophoresis of
DNA fragments separates them based on size only. Using electrophoresis, we can see how many
different DNA fragments are present in a sample and how large they are relative to one another. We
can also determine the absolute size of a piece of DNA by examining it next to a standard "yardstick"
made up of DNA fragments of known sizes.

As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like material. Gels for DNA
separation are often made out of a polysaccharide called agarose, which comes as dry, powdered
flakes. When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will
form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that
are held together by hydrogen bonds and form tiny pores.

At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will
be placed:

Before the DNA samples are added, the gel must be placed in a gel box. One end of the box is
hooked to a positive electrode, while the other end is hooked to a negative electrode. The main body
of the box, where the gel is placed, is filled with a salt-containing buffer solution that can conduct
current. Although you may not be able to see in the image above (thanks to my amazing artistic
skills), the buffer fills the gel box to a level where it just barely covers the gel.

The end of the gel with the wells is positioned towards the negative electrode. The end without wells
(towards which the DNA fragments will migrate) is positioned towards the positive electrode.

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Once the gel is in the box, each of the DNA samples we want to examine (for instance, each PCR
reaction or each restriction-digested plasmid) is carefully transferred into one of the wells. One well is
reserved for a DNA ladder, a standard reference that contains DNA fragments of known lengths.
Commercial DNA ladders come in different size ranges, so we would want to pick one with good
"coverage" of the size range of our expected fragments.

Next, the power to the gel box is turned on, and current begins to flow through the gel. The DNA
molecules have a negative charge because of the phosphate groups in their sugar-phosphate
backbone, so they start moving through the matrix of the gel towards the positive pole. When the
power is turned on and current is passing through the gel, the gel is said to be running.

DNA samples are loaded into wells at negative electrode end of gel.

Power is turned on and DNA fragments migrate through gel (towards the positive electrode).

After the gel has run, the fragments are separated by size. The largest fragments are near the top of
the gel (negative electrode, where they began), and the smallest fragments are near the bottom
(positive electrode).

DNA samples are loaded into wells at negative electrode end of gel.

Power is turned on and DNA fragments migrate through gel (towards the positive electrode).

After the gel has run, the fragments are separated by size. The largest fragments are near the top of
the gel (negative electrode, where they began), and the smallest fragments are near the bottom
(positive electrode).

As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster than longer
ones. After the gel has run for a while, the shortest pieces of DNA will be close to the positive end of
the gel, while the longest pieces of DNA will remain near the wells. Very short pieces of DNA may
have run right off the end of the gel if we left it on for too long (something I've most definitely been
guilty of!).

Once the fragments have been separated, we can examine the gel and see what sizes of bands are
found on it. When a gel is stained with a DNA-binding dye and placed under UV light, the DNA
fragments will glow, allowing us to see the DNA present at different locations along the length of the
gel.

The bp next to each number in the ladder indicates how many base pairs long the DNA fragment is.

A well-defined “line” of DNA on a gel is called a band. Each band contains a large number of DNA
fragments of the same size that have all travelled as a group to the same position. A single DNA
fragment (or even a small group of DNA fragments) would not be visible by itself on a gel.

By comparing the bands in a sample to the DNA ladder, we can determine their approximate sizes.

PCR
Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies
(millions or billions!) of a particular region of DNA. This DNA region can be anything the experimenter
is interested in.

Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used
in some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by
gel electrophoresis, or cloned into a plasmid for further experiments.

PCR is used in many areas of biology and medicine, including molecular biology research, medical
diagnostics, and even some branches of ecology.

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Taq polymerase- Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that
makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used
in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated
(Thermus aquaticus).
T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-stable and
is most active around 70°C (a temperature at which a human or E. coli DNA polymerase would be
nonfunctional). This heat-stability makes Taq polymerase ideal for PCR. As we'll see, high
temperature is used repeatedly in PCR to denature the template DNA, or separate its strands.

PCR primers- Like other DNA polymerases, Taq polymerase can only make DNA if it's given a primer,
a short sequence of nucleotides that provides a starting point for DNA synthesis. In a PCR reaction,
the experimenter determines the region of DNA that will be copied, or amplified, by the primers she or
he chooses.
PCR primers are short pieces of single-stranded DNA, usually around 202020 nucleotides in length.
Two primers are used in each PCR reaction, and they are designed so that they flank the target
region (region that should be copied). That is, they are given sequences that will make them bind to
opposite strands of the template DNA, just at the edges of the region to be copied. The primers bind
to the template by complementary base pairing.

The steps of PCR

The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides
(DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the
enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be
synthesized.

The basic steps are:

Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA strands. This
provides single-stranded template for the next step.

Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences
on the single-stranded template DNA.

Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers,
synthesizing new strands of DNA.

This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2 - 4 hours,
depending on the length of the DNA region being copied. If the reaction is efficient (works well), the
target region can go from just one or a few copies to billions.

That’s because it’s not just the original DNA that’s used as a template each time. Instead, the new
DNA that’s made in one round can serve as a template in the next round of DNA synthesis. There are
many copies of the primers and many molecules of Taq polymerase floating around in the reaction, so
the number of DNA molecules can roughly double in each round of cycling. This pattern of
exponential growth is shown in the image below.

Using gel electrophoresis to visualize the results of PCR

The results of a PCR reaction are usually visualized (made visible) using gel electrophoresis. Gel
electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an
electric current, and it separates DNA fragments according to size. A standard, or DNA ladder, is
typically included so that the size of the fragments in the PCR sample can be determined.

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DNA fragments of the same length form a "band" on the gel, which can be seen by eye if the gel is
stained with a DNA-binding dye. For example, a PCR reaction producing a 400 base pair (bp)
fragment would look like this on a gel:

Left lane: DNA ladder with 100, 200, 300, 400, 500 bp bands.

Right lane: result of PCR reaction, a band at 400 bp.

Left lane: DNA ladder with 100, 200, 300, 400, 500 bp bands.

Right lane: result of PCR reaction, a band at 400 bp.

A DNA band contains many, many copies of the target DNA region, not just one or a few copies.
Because DNA is microscopic, lots of copies of it must be present before we can see it by eye. This is
a big part of why PCR is an important tool: it produces enough copies of a DNA sequence that we can
see or manipulate that region of DNA.

Sanger sequencing
Regions of DNA up to about 900 base pairs in length are routinely sequenced using a method called
Sanger sequencing or the chain termination method.

In the Human Genome Project, Sanger sequencing was used to determine the sequences of many
relatively small fragments of human DNA. The fragments were aligned based on overlapping portions
to assemble the sequences of larger regions of DNA and, eventually, entire chromosomes.

Although genomes are now typically sequenced using other methods that are faster and less
expensive, Sanger sequencing is still in wide use for the sequencing of individual pieces of DNA, such
as fragments used in DNA cloning or generated through polymerase chain reaction (PCR).

Ingredients for Sanger sequencing

Sanger sequencing involves making many copies of a target DNA region. Its ingredients are similar to
those needed for DNA replication in an organism, or for polymerase chain reaction (PCR), which
copies DNA in vitro. They include:

A DNA polymerase enzyme

A primer, which is a short piece of single-stranded DNA that binds to the template DNA and acts as a
"starter" for the polymerase

The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)

The template DNA to be sequenced

However, a Sanger sequencing reaction also contains a unique ingredient:

Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each
labeled with a different color of dye

Dideoxy nucleotides are similar to regular, or deoxy, nucleotides, but with one key difference: they
lack a hydroxyl group on the 3’ carbon of the sugar ring. In a regular nucleotide, the 3’ hydroxyl group
acts as a “hook," allowing a new nucleotide to be added to an existing chain.

Once a dideoxy nucleotide has been added to the chain, there is no hydroxyl available and no further
nucleotides can be added. The chain ends with the dideoxy nucleotide, which is marked with a
particular color of dye depending on the base (A, T, C or G) that it carries.

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The DNA sample to be sequenced is combined in a tube with primer, DNA polymerase, and DNA
nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy
nucleotides are added as well, but in much smaller amounts than the ordinary nucleotides.

The mixture is first heated to denature the template DNA (separate the strands), then cooled so that
the primer can bind to the single-stranded template. Once the primer has bound, the temperature is
raised again, allowing DNA polymerase to synthesize new DNA starting from the primer. DNA
polymerase will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide
instead of a normal one. At that point, no further nucleotides can be added, so the strand will end with
the dideoxy nucleotide.

This process is repeated in a number of cycles. By the time the cycling is complete, it’s virtually
guaranteed that a dideoxy nucleotide will have been incorporated at every single position of the target
DNA in at least one reaction. That is, the tube will contain fragments of different lengths, ending at
each of the nucleotide positions in the original DNA (see figure below). The ends of the fragments will
be labeled with dyes that indicate their final nucleotide.

After the reaction is done, the fragments are run through a long, thin tube containing a gel matrix in a
process called capillary gel electrophoresis. Short fragments move quickly through the pores of the
gel, while long fragments move more slowly. As each fragment crosses the “finish line” at the end of
the tube, it’s illuminated by a laser, allowing the attached dye to be detected.

The smallest fragment (ending just one nucleotide after the primer) crosses the finish line first,
followed by the next-smallest fragment (ending two nucleotides after the primer), and so forth. Thus,
from the colors of dyes registered one after another on the detector, the sequence of the original
piece of DNA can be built up one nucleotide at a time. The data recorded by the detector consist of a
series of peaks in fluorescence intensity, as shown in the chromatogram above. The DNA sequence
is read from the peaks in the chromatogram.

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