POLYMERASE CHAIN REACTION (PCR)

Polymerase chain reaction (PCR) is a molecular biology technique, invented in 1985 by

Kary B. Mullis,[1] for enzymatically replicating DNA without using a living organism,
such as E. coli or yeast. Like amplification using living organisms, the technique allows a
small amount of the DNA molecule to be amplified exponentially. However, because it is
an in vitro technique, it can be performed without restrictions on the form of DNA and it
can be extensively modified to perform a wide array of genetic manipulations.

PCR is commonly used in medical and biological research labs for a variety of tasks,
such as the detection of hereditary diseases, the identification of genetic fingerprints, the
diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA
computing.

Figure 1: A thermal cycler for PCR

PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single
gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy
only short DNA fragments, usually up to 10 kb. Certain methods can copy fragments up
to 47 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell
- for example, a human cell contains about three billion base pairs.

PCR, as currently practised, requires several basic components. These components are:

• DNA template, which contains the region of the DNA fragment to be amplified
 • Two primers, which determine the beginning and end of the region to be
amplified (see following section on primers)
• Taq polymerase (or another durable polymerase), a DNA polymerase, which
copies the region to be amplified
• Deoxynucleotides-triphosphate, from which the DNA Polymerase builds the new
DNA
• Buffer, which provides a suitable chemical environment for the DNA Polymerase

The PCR process is carried out in a thermal cycler. This is a machine that heats and cools
the reaction tubes within it to the precise temperature required for each step of the
reaction. To prevent evaporation of the reaction mixture (typically volumes between 15-
100µl per tube), a heated lid is placed on top of the reaction tubes or a layer of oil is put
on the surface of the reaction mixture. These machines cost more than USD 2,500 in

Primers

The DNA fragment to be amplified is determined by selecting primers. Primers are short,
artificial DNA strands — often not more than 50 and usually only 18 to 25 base pairs
long — that are complementary to the beginning or the end of the DNA fragment to be
amplified. They anneal by adhering to the DNA template at these starting and ending
points, where the DNA polymerase binds and begins the synthesis of the new DNA
strand.

The choice of the length of the primers and their melting temperature (Tm) depends on a
number of considerations. The melting temperature of a primer--not to be confused with
the melting temperature of the DNA in the first step of the PCR process--is defined as the
temperature at which half of the primer binding sites are occupied. The melting
temperature increases with the length of the primer. Primers that are too short would
anneal at several positions on a long DNA template, which would result in non-specific
copies. On the other hand, the length of a primer is limited by the temperature required to
melt it. Melting temperatures that are too high, i.e., above 80°C, can cause problems since
the DNA polymerase is less active at such temperatures. The optimum length of a primer
is generally from 15 to 40 nucleotides with a melting temperature between 55°C and
65°C.

Sometimes degenerate primers are used. These are actually mixtures of similar, but not
identical, primers. They may be convenient if the same gene is to be amplified from
different organisms, as the genes themselves are probably similar but not identical. The
other use for degenerate primers is when primer design is based on protein sequence. As
several different codons can code for one amino acid, it is often difficult to deduce which
codon is used in a particular case. Therefore primer sequence corresponding to the amino
acid isoleucine might be "ATH", where A stands for adenine, T for thymine, and H for
adenine, thymine, or cytosine. (See genetic code for further details about codons.) Use of
degenerate primers can greatly reduce the specificity of the PCR amplification. This
problem can be partly solved by using touchdown PCR.

The three steps in the polymerase chain reaction - the separation of the strands, annealing
the primer to the template, and the synthesis of new strands - take less than two minutes.
Each is carried out in the same vial. At the end of a cycle, each piece of DNA in the vial
has been duplicated.

But the cycle can be repeated 30 or more times. Each newly synthesized DNA piece can
act as a new template, so after 30 cycles, 1 billion copies of a single piece of DNA can be
produced! Taking into account the time it takes to change the temperature of the reaction
vial, 1 million copies can be ready in about three hours.

PCR is valuable to researchers because it allows them to multiply unique regions of DNA
so they can be detected in large genomes. Researchers in the Human Genome Project are
using PCR to look for markers in cloned DNA segments and to order DNA fragments in
libraries.

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR)
The elegant technique of PCR, by which fragments of DNA can be made to replicate very
rapidly, is illustrated.

Figure Legend:
Polymerase chain reaction (PCR), is a common method of creating copies of specific
fragments of DNA. PCR rapidly amplifies a single DNA molecule into many billions of
molecules.

In one application of the technology, small samples of DNA, such as those found in a
strand of hair at a crime scene, can produce sufficient copies to carry out forensic tests.

RAPD stands for Random Amplification of Polymorphic DNA.

RAPD reactions are PCR reactions, but they amplify segments of DNA which are
essentially unknown to the scientist (random).

Often, PCR is used to amplify a known sequence of DNA. Thus, the scientists chooses
the sequence he or she wants to amplify, then designs and makes primers which will
anneal to sequences flanking the sequence of interest. Thus, PCR leads to the
amplification of a particular segment of DNA.

Standard PCR:
However, in RAPD analysis, the target sequence(s) (to be amplified) is unknown. The
scientist will design a primer with an arbitrary sequence. In other words, the scientist
simply makes up a 10 base pair sequence (or may have a computer randomly generate a
10 bp sequence), then synthesizes the primer. The scientist then carries out a PCR
reaction and runs an agarose gel to see if any DNA segments were amplified in the
presence of the arbitrary primer.

Remember! In order for PCR to occur:

• The primers must anneal in a particular orientation (such that they point towards
each other).
• The primers must anneal within a reasonable distance of one another.

In this figure which depicts a RAPD reaction, a large fragment of DNA is used the
template in a PCR reaction containing many copies of a single arbitrary primer.

RAPD’s

Molecular polymorphisms can be identified at random and used for genetic mapping.

Facts

• The complexity of eukaryotic nuclear DNA is sufficiently high that by chance

pairs of sites complementary to single octa- or decanucleotides may exist in the
correct orientation and close enough to one another for PCR amplification.
• With some randomly chosen decanucleotides no sequences are amplified. With
others, the same length products are generated from DNAs of different
individuals. With still others, patterns of bands (such as those illustrated) are not
the same for every individual in a population. The variable bands are commonly
 called random amplified polymorphic DNA (RAPD) bands. Three of the bands in
the diagram are RAPD bands.

RAPD Reaction #1:

• The arrows represent multiple copies of a primer (all primers (arrows) have the
same sequence). The direction of the arrow also indicates the direction in which
DNA synthesis will occur.
• The numbers represent locations on the DNA template to which the primers
anneal.
• Primers anneal to sites 1, 2, and 3 on the bottom strand of the DNA template and
primers anneal to sites 4, 5, and 6 on the top strand of the DNA template.

In this example, only 2 RAPD PCR products are formed:

1) Product A is produced by PCR amplification of the DNA sequence which lies in
between the primers bound at positions 2 and 5.
2) Product B is the produced by PCR amplification of the DNA sequence which lies in
between the primers bound at positions 3 and 6.

Note that no PCR product is produced by the primers bound at positions 1 and 4 because
these primers are too far apart to allow completion of the PCR reaction.

Note that no PCR products are produced by the primers bound at positions 4 and 2 or
positions 5 and 3 because these primer pairs are not oriented towards each other.

Finding Differences Between Genomes Using RAPD Analysis

Consider the figure above.

If another DNA template (genome) was obtained from a different (yet related) source,
there would probably be some differences in the DNA sequence of the two templates.

Suppose there was a change in sequence at primer annealing site #2:

RAPD Reaction #2:

As shown in this figure, the primer is no longer able to anneal to site #2, and thus the
PCR product A is not produced. Only product B is produced.

If you were to run the 2 RAPD PCR reactions diagramed above on an agarose gel, this is
what you would see:
RAPD-PCR is a wide-spread method for genetic fingerprinting of eukaryotes. Its major
advantage is that absolutely no sequence information of the target genome is required.

Essentially all RAPD-Primers will give you a characteristic fingerprint of any target
genome. Band numbers normally range between 2 and 10.

If fingerprinting is all you need, say for unequivocal recognizing natural isolates of a
microorganism, you need not play around too much. Application of 3-6 random primers
will normally do.

If you want to see correlations between RAPD band patterns and defined traits of your
target organism some more efforts are required. A good advice is to start with 20
different primers and something like 6 target organisms of every phenotypic group that
you need to characterize.

You may use any oligonucleotide as RAPD primer above a chain length of 9. It is
essentially the annealing temperature of the PCR that defines the pattern, not so much the
length of the primer.

For generating random sequences of a given chain length, you may use the 'RAPD-
generator' in the Java applet on this page, written by Gebhard Wöstemeyer. Do not rely
on imagination - sequences by the 'RAPD-generator' are much more random than your
own.

Good results were obtained with the following random primers for fungal DNA:
primer 6: 5´-GAAACAGCGG-3´
primer 8: 5´-GGAGCCCAC-3´
primer 14: 5´-GCCGTCTACG-3´
primer 17: 5´-GGCATCGGCC-3´
primer 21: 5´-GTGAGCGTC-3´

RAPD amplification mixtures contain in 50 µl

• approximately 25 to 50 ng of genomic DNA

• 40 ng nona- or decamer primer
• 16 mM (NH4)2SO4
• 50 mM Tris-HCl pH 8.8 (at 25 °C)
• 0.1% Tween 20
• 0.2 mM of each dNTP
• 3 mM magnesium chloride
• 1 U Taq Polymerase

We use the following temperature profile:

• initial denaturation step: 5 min -95 °C

• 30 cycles of:
primer annealing: 60 s - 32 °C
primer extension: 20 s - 72 °C
DNA denaturation: 20 s - 95 °C
• cool down to 20 °C

Amplicons are separated electrophoretically in 1.2 % agarose gels in 1 x TAE or for

higher resolution in 5 % native polyacrylamide gels (5% w/v acrylamide, 0.17% w/v
bisacrylamide, 0.08% w/v ammonium peroxodisulfate, 0.05% v/v TEMED) in 1 x TBE
buffer for 1.5 h at 8 V/cm.

RAPD analysis was done to determine intraspecific variability in Andrographis

paniculata, a popular antipyretic and hepatoprotective drug used in traditional medicine in
India. The accessions collected from parts of India and south-east Asia on molecular
analysis revealed moderate variation within the species. Similarity measurement using
UPGMA followed by cluster analysis resulted in 5 major groups based on geographical
distribution that generally reflected expected trends between the genotypes. There were
also important exceptions like AP-48, an accession from Thailand showing close
resemblance to AP-38 collected from Tamil Nadu and AP-29 from Assam significantly
diverse from the rest of the native genotypes. The results indicated that RAPD could be
effectively used for genetic diversity analysis in wild species of prospective value as it is
reliable, rapid and superior to those based on pedigree information.