PCR

 Polymerase chain reaction, or PCR, is a technique to make many copies

of a specific DNA region in vitro (in a test tube rather than an organism).

 PCR relies on a thermostable DNA polymerase, Taq polymerase, and

requires DNA primers designed specifically for the DNA region of
interest.

 In PCR, the reaction is repeatedly cycled through a series of temperature

changes, which allow many copies of the target region to be produced.

 PCR has many research and practical applications. It is routinely used in

DNA cloning, medical diagnostics, and forensic analysis of DNA.

What is 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.
For example, it might be a gene whose function a researcher wants to
understand, or a genetic marker used by forensic scientists to match crime
scene DNA with suspects.

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.

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).

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.

Template DNA:

5' TATCAGATCCATGGAGT...GAGTACTAGTCCTATGAGT 3' 3'

ATAGTCTAGGTACCTCA...CTCATGATCAGGATACTCA 5'

Primer 1: 5' CAGATCCATGG 3' Primer 2:

When the primers are bound to the template, they can be extended by the
polymerase, and the region that lies between them will get copied.
 [More detailed diagram showing DNA and primer directionality]

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:

1. Denaturation (96 °\text C96°C96, °, start text, C, end text): Heat the
reaction strongly to separate, or denature, the DNA strands. This provides
single-stranded template for the next step.

2. Annealing (555555 - 656565°\text C°C°, start text, C, end text): Cool the
reaction so the primers can bind to their complementary sequences on the
single-stranded template DNA.

3. Extension (72 °\text C72°C72, °, start text, C, end text): Raise the reaction
temperatures so Taq polymerase extends the primers, synthesizing new
strands of DNA.

This cycle repeats 252525 - 353535 times in a typical PCR reaction, which
generally takes 222 - 444 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.

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 400400400 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.

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.

Applications of PCR
Using PCR, a DNA sequence can be amplified millions or billions of
times, producing enough DNA copies to be analyzed using other
techniques. For instance, the DNA may be visualized by gel
electrophoresis, sent for sequencing, or digested with restriction enzymes
and cloned into a plasmid.

PCR is used in many research labs, and it also has practical applications in
forensics, genetic testing, and diagnostics. For instance, PCR is used to
amplify genes associated with genetic disorders from the DNA of patients
(or from fetal DNA, in the case of prenatal testing). PCR can also be used
to test for a bacterium or DNA virus in a patient's body: if the pathogen is
present, it may be possible to amplify regions of its DNA from a blood or
tissue sample.
RNA Isolation Protocol
Total RNA isolation from Bacterial Cells
Principle of RNA Isolation
Total RNA is isolated and separated from DNA and protein after extraction with a solution
called as Trizol. Trizol is an acidic solution containing guanidinium thiocyanate (GITC),
phenol and chloroform. GITC irreversibly denatures proteins and RNases. This is followed
by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase,
while most of DNA and proteins remain either in the interphase or in the lower organic
phase. Total RNA is then recovered by precipitation with isopropanol. RNase enzymes can
be inactivated by including diethyl pyrrocarbonate (DEPC).

Materials Required for RNA Isolation

 Bacterial culture
 Trizol
 Chloroform
 Isopropanol solution
 TAE buffer
 70% ethanol
Procedure of RNA Isolation
1. Take 800 μL of bacterial culture in a fresh eppendorf.
2. To this add 160 μL of Trizol (1/5th of culture volume).
3. The solution was mixed well by pipetting several times.
4. To this add 32 μl of chloroform (1/5th volume of trizol).
5. Incubate for 2 to 5 minutes and centrifuge at 12000 rpm for 15 minutes at 4° C
6. Transfer the aqueous phase into a new tube and add equal volume of isopropanol. Mix
well.
7. Centrifuge at 10000 rpm for 10 minutes at 4° C.
8. Discard the supernatant and resuspend the pellet in 70% ethanol. • Again centrifuge at
10000 rpm for 10 minutes at 4° C.
9. Discard the supernatant.
10. Air dry the pellet at 37° C for 10-15 minutes.
11. Resuspend the pellet in 50 μL of TE buffer.
12. Analyse the RNA sample quantitatively and qualitatively.
Isolation of RNA from Animal Cells or Tissues
Materials Reagents
 TRIzol reagent
 Chloroform
Instruments
 Fume hood
 Vortexer
 Micropipettes
 Chilled microcentrifuge
 Pellet pestle homogenizer
Procedure
1. Clean the workstation (pipettor shaft, benchtop) with a surface decontamination
solution that destroys RNases.
2. Chill the microcentrifuge to 4 ºC.
3. Homogenization fresh tissue or cell culture:
 Fresh tissue is preferable for optimal RNA isolation. Alternatively, tissue should be
submerged in RNA stabilization reagent immediately after dissection and stored at –
80 °C.
 Add 1 ml TRIzol reagent per 100 mg fresh tissue, mince on ice using sterile scalpels,
and homogenize with a sterile pellet pestle probe.
 In case of cell cultures, they should be processed immediately after removal from the
incubator.
 Either centrifuge cells grown in suspension at 300 x g for 5 min at RT (15-25 ºC) and
discard supernatant or remove the culture medium from cells grown in monolayer.
 Add 1 ml TRIzol reagent per 1 × 107 cells to cell pellets or directly to the culture dish
or flask for cells grown in monolayer. Resuspend the lysate with a sterile, disposable
1-ml pipette tip.
4. Transfer the tissue or cell lysate to a 2-ml siliconized low-retention tube.
5. Pass sample through sterile, disposable 21 g needle 10 times. In doing so, high-
molecular weight cellular components (DNA) are fragmented, thus minimizing their
presence in the aqueous phase.
6. Let homogenate sit at RT (15-25 ºC) for 5 min for complete dissociation of
nucleoprotein complexes.
7. Let homogenate sit at RT (15-25 ºC) for 3 min.
 8. Centrifuge at 12,000 x g for 15 min at 4°C. After centrifugation, heat the centrifuge to
room temperature (15–25°C).
9. Transfer upper aqueous phase (600 ul) to new 1.5 ml RNase-free tube.
10. Proceed to alcohol precipitation.
11. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use
0.5 ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the initial
homogenization.
12. Incubate samples at 15 to 30oC for 10 minutes and centrifuge at not more than 12,000
x g for 10 minutes at 2 to 4oC.
13. Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol,
adding at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial
homogenization.
14. Mix the samples by vortexing and centrifuge at no more than 7,500 x g for 5 minutes
at 2 to 8 oC. Repeat.
15. Air-dry or vacuum dry RNA pellet for 5-10 minutes.
16. Dissolve RNA in DEPC-treated water by passing solution a few times through a
pipette tip.
17. Subject to spectrophotometric analysis to determine sample concentration and purity.
Expected Results
 A gel-like pellet on the side and bottom of the tube of RNA precipitate.
 On taking OD at 260 nm and 280 nm, the A260/A280 ratio should be above 1.6.

DNA Etraction and Demonstration

In order to study DNA, you first have to get it out of the cell. In eukaryotic cells, such
as human and plant cells, DNA is organized as chromosomes in an organelle called
the nucleus. Bacterial cells have no nucleus. Their DNA is organized in rings or
circular plasmids, which are in the cytoplasm. The DNA extraction process frees
DNA from the cell and then separates it from cellular fluid and proteins so you are
left with pure DNA.

The three basic steps of DNA extraction are 1) lysis, 2) precipitation, and 3)
purification.

Step 1: Lysis
In this step, the cell and the nucleus are broken open to release the DNA inside and
there are two ways to do this. First, mechanical disruption breaks open the cells. This
can be done with a tissue homogenizer (like a small blender), with a mortar and
pestle, or by cutting the tissue into small pieces. Mechanical disruption is particularly
important when using plant cells because they have a tough cell wall.
Second, lysis uses detergents and enzymes such as Proteinase K to free the DNA
and dissolve cellular proteins.
Step 2: Precipitation
When you complete the lysis step, the DNA has been freed from the nucleus, but it is
now mixed with mashed up cell parts. Precipitation separates DNA from this cellular
 debris. First, Na+ ions (sodium) neutralize the negative charges on the DNA
molecules, which makes them more stable and less water soluble. Next, alcohol
(such as ethanol or isopropanol) is added and causes the DNA to precipitate out of
the aqueous solution because it is not soluble in alcohol.
Step 3: Purification
Now that DNA has been separated from the aqueous phase, it can be rinsed with
alcohol to remove any remaining unwanted material and cellular debris. At this point
the purified DNA is usually re-dissolved in water for easy handling and storage.

DEMONSTRATION OF THE ISOLATED DNA

 Gel electrophoresis is a technique used to separate DNA fragments
according to their size.

 DNA samples are loaded into wells (indentations) at one end of a gel, and
an electric current is applied to pull them through the gel.

 DNA fragments are negatively charged, so they move towards the positive
electrode. Because all DNA fragments have the same amount of charge
per mass, small fragments move through the gel faster than large ones.

 When a gel is stained with a DNA-binding dye, the DNA fragments can be
seen as bands, each representing a group of same-sized DNA fragments.

Introduction
Suppose you have just done a PCR reaction, making many copies of a
target DNA region. Or perhaps you’ve done some DNA cloning, trying to
"paste" a gene into a circular DNA plasmid.

Now, you want to check and see whether your PCR worked, or whether
your plasmid has the right gene in it. What technique can you use to
visualize (directly observe) the fragments of DNA?

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.
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.

How do DNA fragments move through the

gel?
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).
Based on similar diagram in Reece et al.^22squared
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 awhile, 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!).

Visualizing the DNA fragments

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
traveled 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. For instance, the bright band on the gel above is
roughly 700700700 base pairs (bp) in size.