PCR, or Polymerase Chain Reaction

First popularly thought to have been conceived by Dr. Kerry Mullis in 1983 while working at the
Cetus Corporation in Emeryville, CA, along with other researchers Cetus Corporation discovered
a method to start and stop DNA polymerase enzyme activity at specific points along a single
strand of DNA. On the other hand, some pioneering research was also done by Gobind Khorana,
who described a basic principle of replicating a piece of DNA using two primers (1971). Kerry
Mullis discovered that by harnessing this component of molecular reproduction technology, a
target DNA of interest could be amplified exponentially. This DNA amplification procedure was
an in vitro process (meaning in a test-tube).

Cell-free DNA amplification by PCR was able to simplify many of the standard procedures for
DNA cloning, DNA analysis, and the modification of DNA. Previous molecular biology
techniques for isolating a specific piece of DNA had relied on gene cloning, which is a tedious
and slow procedure. PCR, as Kerry Mullis stated “lets you pick the piece of DNA you’re
interested in and have as much of it as you want”.

Progress for the development was initially limited by primer synthesis and polymerase
purification issues. When Cetus scientists eventually succeeded in making the polymerase chain
reaction perform as desired in a reliable fashion, they had an immensely powerful technique for
providing almost unlimited quantities of the precise genetic material molecular biologists and
others required for their research. Since the first report in1985, more than 5000 scientific papers
were published by 1992.

Polymerase Chain Reaction: An Alternative to cloning

An alternative to cloning called the polymerase chain reaction PCR can be used to directly
amplify rare specific DNA sequences in a complex mixture when the ends of the sequence are
known. This method of amplifying rare sequences from a mixture has numerous applications in
basic research, human genetics testing and forensics.

The PCR process consists of the following steps

o Initialization. The mixture is heated at 96°C for 5 minutes to ensure that the DNA strands
as well as the primers have melted
o Melting, where it is heated at 96°C for 30 seconds
o Annealing by heating at 68°C for 30 seconds
o Elongation by heating 72°C for 45 seconds
Genomic DNA is digested into large fragments using a restriction enzyme and then is heat-
denatured into single strands. Two synthetic oligonucleotides complementary to the 3’ ends of
the target DNA segment of interest are added in great excess to the denatured DNA, and the
temperature is lowered to 50-60° C.

o The genomic DNA remains denatured, because the complementary strands are at
too low a concentration to encounter each other during the period of incubation,
but the specific oligonucleotides, which are at a very high concentration,
hybridize with their complementary sequences in the genomic DNA.
o The hybridized oligonucleotides then serve as primers for DNA chain synthesis,
which begins upon addition of a supply of deoxynucleotides and a temperature
resistant DNA polymerase such as that from Thermus aquaticus (a bacterium that
lives in hot springs).
o This enzyme, Taq polymerase can extend the primers at temperatures up to 72°C.
o When synthesis is complete, the whole mixture is heated up further (to 95°) to
melt the newly formed DNA duplexes.
o When the temperature is lowered again, another round of synthesis takes place
because excess primer is still present.
o Repeated cycles of synthesis (cooling) and melting (heating) quickly amplify the
sequence of interest.
o At each round, the number of copies of the sequences between the primer sites is
doubled; therefore, the desired sequence increases exponentially.

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

• DNA template that contains the region of the DNA fragment to be amplified
One or more primers , which are complementary to the DNA regions at the 5' and 3' ends of the DNA
region that is to be amplified (see following section on primers)
• Taq Ploymerase enzyme (or another DNA polymerase with a temperature optimum at around
70°C), a Dna Polymerase , used to synthesize a DNA copy of the region to be amplified
• Deoxyribo neucletiode , (dNTPs) from which the DNA polymerase builds the new DNA
• Buffer Solution , which provides a suitable chemical environment for optimum activity and
stability of the DNA polymerase
• Divalent cation , Mg+ , Mn++_ : ions; generally Mg2+ is used, but Mn2+ can be utilized for
PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA
synthesis
• Monovalent cation K+ ions
Conditions and Factors Affecting PCR : Factors Affecting PCR

PCR Reaction Volume

Increasing the PCR reaction volume proportionally to a PCR reaction that is optimized or works
well, in general there will be an increase in the amount of product with increasing volume.
Exceptions to this include older mineral oil PCR machines and PCR thermocyclers without a
heated-lid. In such cases, increasing the PCR reaction volume will negatively affect the PCR
product and efficiency of the reaction.

PCR Primer Design

Primer are usually chosen to be between 18-25 nucleotides in length. Longer primers also work
30-40 nucleotides or bp during pcr

A GC clamp at the 5' ends of the primers helps.

One can thus design primers with 1 or 2 nucleotides GC at the start and ends of the primers if
possible

Types of PCR

• AFLP PCR
• Alu PCR
• Colony PCR
• Hot Start PCR
• Inverse PCR
• In Situ PCR
• Long PCR
• Reverse Transcriptase PCR
• Real Time PCR
• Standard PCR
General PCR Procedure

Figure 1: pcr reaction and than after heating and cooling goes on and on .
PCR Operating Problems / Trouble shooting :

• Long Non-specific Products in PCR

• PCR Primer Dimers
• PCR Contamination

1) Long Non-specific Products in PCR:When running an agarose gel we spot larger

non-specific PCR bands that are not the right size.

Solutions to Long Non-specific Products in PCR:

o Decrease annealing time

o Decrease extension time and temperature to 62-68º C
o Use less primer and less Taq polymerase
o Take less DNA template

2) PCR Primer Dimers: When running an agarose gel, we spot some very small PCR
bands. These are the size of primer or about the size of two your primers (suppose A and B)
together. These are termed "primer dimers" and are formed by the annealing of primer with
itself or with the other primer.

Hence the Primer dimers are caused by lack of binding of the primers to the template due to
inefficient primer annealing to the template due to Higher affinity binding of the primers to each
other or themselves also causes primer dimers. Primer dimers are seen as major band which
migrates at less than 100bp on an agarose gel.

Solutions to primer dimers in PCR:

o Use less primer and optimize annealing conditions

o Re-design primer which should have lower self-annealing or self-hybridization potential
o Conduct PCR with and without formamide.
o Increase DNA template amount (concentration).
o Increase annealing temperature (try optimizing using a gradient PCR machine to find optimal
temperature for annealing).
o Try using HotStart PCR instead of regular Taq polymerase PCR.

3) PCR Contamination: PCR contamination is caused by DNA samples which

Solutions to PCR contamination:

o Use Zero Aerosol Pipette Tips.

o Preparing fresh new autoclaved water
PCR Limitations and Disadvantages:

• The High Sensitivity of PCR

• Polymerase Errors
• PCR Polymerase Enzyme Proficiency and PCR Product Size / Length
Limitations

The High Sensitivity of PCR: Due to the extremely high sensitivity of PCR, contamination
from non-template PCR present in the lab environment (from bacteria, viruses, and your own
DNA) presents a real problem. Precautions therefore must be taken.

Several steps can be taken to prevent and minimize PCR contamination including the use of
aerosol barrier tip filters, laminar flow cabinet, and a special preparation area for PCR separate
from the DNA isolation area.

Polymerase Errors : DNA polymerase are not perfect by any means, however they are quite
good at what they do. Mistakes and errors do happen in PCR by DNA polymerases in the test
tube and in the human body. The polymerases used in PCR often lack 3' to 5' exonuclease
activity such as Taq polymerase. This enzyme lacks the ability to correct misincorporated
nucleotides, an activity which is present in higher eukaryotes.

A polymerase lacking the 3' to 5' exonuclease activity has a higher error rate, around 1 in 10000
bases are misincorporated.

Recombinant polymerases have been generated and other polymerases have been isolated which
are available to more accurate PCR and have a variety of applications such as sequencing.

Examples of polymerases with 3' to 5' exonuclease activity include: Vent, which is extracted
from Thermococcus litoralis , Pfu which is extracted from Pyrococcus furiosus, and Pwo which
is extracted from Pyrococcus woes

PCR Polymerase Enzyme Proficiency and PCR Product Size / Length Limitations : DNA
polymerase are proficient enough to efficiently amplify DNA products up to a few thousand
basepairs (2-5 kb). PCRs of longer products are less efficient due to enzyme activity loss, and
inaccuracies introduced by longer PCRs. Adding fresh DNA polymerase helps with enzyme
activity lost due to the half life of the polymerase, however this does not help when accurate PCR
is required.

It is possible to amplify PCR products up to 20kb using lower or slower heating cycles and
special mixtures of polymerases. Long and Accurate PCR is accomplished by using mixtures of
processive polymerases and high fidelity Polymerases

Need for target DNA sequence information

In order to construct specific oligonucleotide primers that permit selective amplification of a

particular DNA sequence, some prior sequence information is necessary. This normally means
that the DNA region of interest has been partly characterized previously, often following cell-
based DNA cloning. However, a variety of techniques have been developed that reduce or even
exclude the need for prior DNA sequence information concerning the target DNA, when certain
aims are to be met. For example, previously uncharacterized DNA sequences can sometimes be
cloned using PCR with degenerate oligonucleotides if they are members of a gene or repetitive
DNA family at least one of whose members has previously been characterized. In some cases,
PCR can be used effectively without any prior sequence information concerning the target DNA
to permit indiscriminate amplification of DNA sequences from a source of DNA that is present
in extemely limited quantities . PCR can be applied to ensure whole genome amplification, it
does not have the advantage of cell-based DNA cloning in offering a way of separating the
individual DNA clones comprising a genomic DNA lib

PCR optimization

Since PCR is very sensitive, i.e., requiring only a few DNA molecules for amplification across several
orders of magnitude, adequate measures to avoid contamination from any DNA present in the lab
environment ( viruses, lab staff's skin , bacteria etc.) should be taken. Thus DNA sample preparation,
reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis,
should all be performed in separate areas.

In practice, one area should be dedicated to reaction assembly before the PCR and another area to post-
PCR processing, such as electrophoresis or purification of PCR products. For the preparation of reaction
mixtures, a laminar flow cabinet with UV lamp is recommended, and pipettes with filter tips should be
used. Fresh gloves should be used for each PCR step as well as displacement pipettes with aerosol filters.
The reagents for PCR should be prepared separately and used solely for this purpose. Aliquots should be
stored separately from other DNA samples. A control reaction, omitting template DNA (also called
negative control), should always be performed alongside experimental PCRs, to check for possible
contamination of reagents with extraneous DNA or for primer multimer formation.

Hairpins

DNA secondary structure , caused by base-pairing between nucleotides on the same strand of the
molecule, can cause folding or even knotting of the DNA template or the primers, leading to decreased
yield or total failure of the reaction. Hairpins, direct folding of the DNA caused by a run of
complementary bases or an inversion, are the most common problems of this sort.

Typically, this calls for choosing different primers; secondary structures in the template DNA are not as
serious as those in the primers, as the DNA polymerase will "flatten out" most secondary structures
( unless they are particularly robust.) template .

However, if use of hairpin-forming primers is necessary, as may be the case in PCR splicing and cloning,
the problem can be ameliorated somewhat by use of DMSO or Glycerol Templalate (These chemicals
can be added to the PCR mastermix to interrupt secondary structures).
Advantages of PCR as a cloning method

Speed and ease of use : DNA cloning by PCR can be performed in a few hours, using relatively
unsophisticated equipment. Typically, a PCR reaction consists of 30 cycles containing a de
naturation , synthesis and reannealing step, with an individual cycle typically taking 3–5 min in
an automated thermal cycler. This compares favorably with the time required for cell-based
DNA cloning, which may take weeks. Clearly, some time is also required for designing and
synthesizing oligonucleotide primers, but this has been simplified by the availability of computer
software for primer design and rapid commercial synthesis of custom oligonucleotides. Once
the conditions for a reaction have been tested, the reaction can then be repeated simply.

Sensitivity: PCR is capable of amplifying sequences from minute amounts of target DNA, even
the DNA from a single cell Such exquisite sensitivity has afforded new methods of studying
molecular pathogenesis and has found numerous applications in forensic science, in diagnosis, in
genetic linkage analysis using single-sperm typing and in molecular paleontology studies, where
samples may contain minute numbers of cells. However, the extreme sensitivity of the method
means that great care has to be taken to avoid contamination of the sample under investigation by
external DNA, such as from minute amounts of cells from the operator.

Robustness: PCR can permit amplification of specific sequences from material in which the
DNA is badly degraded or embedded in a medium from which conventional DNA isolation is
problematic. As a result, it is again very suitable for molecular anthropology and paleontology
studies, for example the analysis of DNA recovered from archaeological remains. It has also
been used successfully to amplify DNA from formalin-fixed tissue samples, which has important
applications in molecular pathology and, in some cases, genetic linkage

Applications or Uses of PCR

PCR can be used for a broad variety of experiments and analyses. Some examples are discussed
below.
Genetic fingerprinting

Genetic fingerprinting is a forensic technique used to identify a person by comparing his or her
DNA with a given sample. An example is blood from a crime scene being genetically compared
to blood from a suspect. The sample may contain only a tiny amount of DNA (obtained from a
source such as blood, semen, saliva, hair, or other organic material). Theoretically, just a single
strand is needed. First, one breaks the DNA sample into fragments using restriction enzymes;
then amplifies them using PCR. The amplified fragments are then separated using gel
electrophoresis. The overall layout of the DNA fragments is called a DNA fingerprint. Since
there is a very tiny possibility that two individuals may have the same sequences (one in several
million), the technique is more effective at acquitting a suspect than proving the suspect guilty.

Paternity testing
Detection of hereditary diseases

The detection of hereditary diseases in a given genome is a long and difficult process, which can
be shortened significantly by using PCR. Each gene in question can easily be amplified through
PCR by using the appropriate primers and then sequenced to detect mutations.

Viral diseases, too, can be detected using PCR through amplification of the viral DNA. This
analysis is possible right after infection, which can be from several days to several months before
actual symptoms occur. Such early diagnoses give physicians a significant lead in treatment.

Cloning genes

Cloning a gene, not to be confused with cloning a whole organism, describes the process of
isolating a gene from one organism and then inserting it into another organism (now termed a
genetically modified organism (GMO)). PCR is often used to amplify the gene, which can then
be inserted into a vector (a vector is a piece of DNA which 'carries' the gene into the GMO) such
as a plasmid (a circular DNA molecule) (Fig. 5). The DNA can then be transferred into an
organism (the GMO) where the gene and its product can be studied more closely. Expressing a
cloned gene (when a gene is expressed the gene product (usually protein or RNA) is produced by
the GMO) can also be a way of mass-producing useful proteins, for example medicines or the
enzymes in biological washing powders. The incorporation of an affinity tag on a recombinant
protein will generate a fusion protein which can be more easily purified by affinity
chromatography
Analysis of ancient DNA

Using PCR, it becomes possible to analyze DNA that is thousands of years old. PCR techniques have
been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on
human DNA, in applications ranging from the analysis of Egyptian mummies to the identification
of a Russian Tsar.

Genotyping of specific mutations

Through the use of allele-specific PCR, one can easily determine which allele of a mutation or
polymorphism an individual has. Here, one of the two primers is common, and would anneal a short
distance away from the mutation, while the other anneals right on the variation. The 3' end of the allele-
specific primer is modified, to only anneal if it matches one of the alleles. If the mutation of interest is a T
or C single nucleotide polymorphism (T/C SNP), one would use two reactions, one containing a primer
ending in T, and the other ending in C. The common primer would be the same. Following PCR, these
two sets of reactions would be run out on an agarose gel, and the band pattern will tell you if the
individual is homozygous T, homozygous C, or heterozygous T/C. This methodology has several
applications, such as amplifying certain haplotypes (when certain alleles at 2 or more SNPs occur
together on the same chromosome Linkage Disequilibrium) or detection of recombinant chromosomes
and the study of meiotic recombination.

Comparison of gene expression

Researchers have used traditional PCR as a way to estimate changes in the amount of a gene's
expression. Ribonucleic acid (RNA) is the molecule into which DNA is transcribed prior to making a
protein, and those strands of RNA that hold the instructions for protein sequence are known as messenger
RNA (mRNA). Once RNA is isolated it can be reverse transcribed back into DNA (complementary
DNA to be precise, known as c DNA), at which point traditional PCR can be applied to amplify the gene,
this methodology is called RT-PCR. In most cases if there is more starting material (mRNA) of a gene
then during PCR more copies of the gene will be generated. When the products of the PCR process are
run on an agarose gel (see Figure 3 above) a band, corresponding to a gene, will appear larger on the gel
(note that the band remains in the same location relative to the ladder, it will just appear fatter or
brighter). By running samples of amplified c DNA from differently treated organisms one can get a
general idea of which sample expressed more of the gene of interest. A quantative RT-PCR method has
been developed, it is called Real-time PCR .