Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction
(PCR)
• PCR is a means to amplify a particular piece of DNA
• Amplify= making numerous copies of a segment of
DNA
• PCR can make billions of copies of a target sequence
of DNA in a few hours
• PCR Polymerase chain reaction (PCR) is a technique
used in molecular biology to amplify a single copy or a
few copies of a segment of DNA across several orders of
magnitude, generating thousands to millions of copies of
a particular DNA sequence.
History
• Developed in 1983 by Kary Mullis, PCR is now a
common technique used in clinical and research
laboratories for a broad variety of applications.
• In 1993, Mullis was awarded the Nobel Prize in
Chemistry for his work on PCR.
• Its applications are vast and PCR is now an
integral part of Molecular Biology
History
• 1985: First publication of PCR by Cetus
Corporation appears in Science.
• 1986: Purified Taq polymerase is first used
in PCR
• 1988: PerkinElmer introduces the
automated thermal cycler.
• 1989: Science declares Taq polymerase
"molecule of the year.
DNA Replication vs. PCR
• PCR is a laboratory version of DNA
Replication in cells
• The laboratory version is commonly called
“in vitro” since it occurs in a test tube while
“in vivo” signifies occurring in a living cell.
DNA Replication in Cells (in vivo)
• DNA replication is the copying of
DNA
• It typically takes a cell just a few
hours to copy all of its DNA
• DNA replication is semi-conservative
(i.e. one strand of the DNA is used
as the template for the growth of a
new DNA strand)
• This process occurs with very few
errors (on average there is one error
per 1 billion nucleotides copied)
• More than a dozen enzymes and
proteins participate in DNA
replication
Purpose of PCR
• To amplify a lot of double-stranded DNA
molecules (fragments) with same (identical)
size and sequence by enzymatic method and
cycling condition.
• For instance, DNA amplified by PCR may
be sent for sequencing, visualized by gel
electrophoresis, or cloned into a plasmid
for further experiments.
Components of PCR
• A basic PCR set-up requires several
components and reagents, including:
• 1. DNA template:
• DNA template is DNA target sequence.
• DNA template is the DNA molecule that contains
the DNA region (segment) to be amplified, the
segment we are concerned which is the target
sequence.
2. DNA polymerase:
• DNA polymerase sequentially adds nucleotides
complimentary to template strand at 3’-OH of the
bound primers and synthesizes new strands of
DNA complementary to the target sequence.
• The most commonly used DNA polymerase is

Taq DNA polymerase (from Thermus aquaticus,
a thermophillic bacterium) because of high
temperature stability.
• Pfu DNA polymerase (from Pyrococcus furiosus)
is also used widely because of its higher fidelity
(accuracy of adding complimentary nucleotide).
• Mg2+ ions in the buffer act as co-factor for DNA

polymerase enzyme and hence are required for
the reaction.
3. Primers
• Primers are synthetic DNA strands of
about 18 to 25 nucleotides complementary
to 3’ end of the template strand.
• DNA polymerase starts synthesizing new
DNA from the 3’ end of the primer
• Two primers must be designed for PCR; the
forward primer and the reverse primer.
• The forward primer is complimentary to the 3’
end of antisense strand (3’-5’)
• The reverse primer is complimentary to the 3’
end of sense strand (5’-3’).
• If we consider the sense strand (5’-3’) of a gene,
for designing primers, then forward primer is the
beginning of the gene and the reverse primer is
the reverse-compliment of the 3’ end of the
gene.
Primer Design
• Primer Design Primers should bind to template
with good specificity and strength.
• If primers do not bind to correct template, wrong
sequence will get amplified.
• Optimal primer sequences and appropriate
primer concentrations are essential for maximal
specificity and efficiency in PCR.
4. Nucleotides
• dNTPs or deoxynucleotide triphosphates:
• All types of nucleotides are "building
blocks" for new DNA strands and essential
for reaction.
• It includes Adenine(A), Guanine(G),
Cytosine(C), Thymine(T) or Uracil(U).
5. Buffer
• A Buffer solution providing a suitable
chemical environment for optimum activity
and stability of DNA polymerase.
6. Bivalent cations
• Bivalent cations in the form of MgCl2,
typically Magnesium (Mg) or manganese
(Mn) ions; Mg2+ is the most commonly
used as a co-factor that catalyzes the
activity of Taq polymerase,
• but Mn2+ can be used for PCR-mediated
DNA mutagenesis, as a higher Mn2+
concentration increases the error rate
during DNA synthesis.
7. Monovalent cations
• Monovalent cations in the form of KCl,
typically potassium (K) ions that facilitate
bonding between primer and template
strand.
Procedure
• There are three major steps in a PCR,
which are repeated for 30 or 40 cycles.
• This is done on an automated cycler,
which can heat and cool the tubes with the
reaction mixture in a very short time
1. Denaturation at 94°C :
• During the heating step (denaturation), the
reaction mixture is heated to 94°C for 1
min, which causes separation of DNA
double stranded. Now, each strand acts as
template for synthesis of complimentary
strand.
2. Annealing at 54°C :
• This step consist of cooling of reaction
mixture after denaturation step to 54°C,
which causes hybridization (annealing) of
primers to separated strand of DNA
(template).
• The length and GC-content (guanine-
cytosine content) of the primer should be
sufficient for stable binding with template.
• Guanine pairs with cytosine with three hydrogen
bonding adenine binds with thymine with two
hydrogen bonds.
• Thus, higher GC content results in stronger
binding.
• In case GC content is less, length may be
increased to have stronger binding (more
number of H bonding between primer and
template).
3. Extension at 72°C :
• The reaction mixture is heated to 72°C
which is the ideal working temperature for
the Taq polymerase. The polymerase adds
nucleotide (dNTP's) complimentary to
template on 3’ –OH of primers thereby
extending the new strand.
The DNA of interest is amplified by
a power of 2 for each PCR cycle
For example, if you subject your DNA of interest to 5 cycles of
PCR, you will end up with 25 (or 64) copies of DNA.
Similarly, if you subject your DNA of interest to 40 cycles of

PCR, you will end up with 240 (or ) copies of DNA!
PCR has become a very powerful
tool in molecular biology
• One can start with a single sperm cell or stand of
hair and amplify the DNA sufficiently to allow for
DNA analysis and a distinctive band on an
agarose gel.
• One can amplify fragments of interest in an
organism’s DNA by choosing the right primers.
• One can use the selectivity of the primers to
identify the likelihood of an individual carrying a
particular allele of a gene.
PCR and Disease
• Primers can be created that will only bind and amplify
certain alleles of genes or mutations of genes
• This is the basis of genetic counseling and PCR is used as
part of the diagnostic tests for genetic diseases.
• Some diseases that can be diagnosed with the help of
PCR:
• Huntington's disease
• cystic fibrosis
• Human immunodeficiency virus
Huntington’s Disease (HD)
• HD is a genetic disorder characterized by abnormal body
movements and reduced mental abilities
• HD is caused by a mutation in the Huntingtin (HD) gene
• In individuals with HD, the HD gene is “expanded”
– In non-HD individuals, the HD gene has a pattern called trinucleotide
repeats with “CAG” occurring in repetition less than 30 times.
– IN HD individuals, the “CAG” trinucleotide repeat occurs more that 36
times in the HD gene
• PCR can be performed on an individual’s DNA to determine whether
the individual has HD.
– The DNA is amplified via PCR and sequenced (a technique by which
the exact nucleotide sequence is determined) and the number of
trinucleotide repeats is then counted.
Cystic Fibrosis (CF)
• CF is a genetic disease characterized by severe breathing
difficulties and a predisposition to infections.
• CF is caused by mutations in the cystic fibrosis transmembrane
conductance regulator (CTFR) gene.
• In non-CF individuals, the CTFR gene codes for a protein that is a
chloride ion channel and is involved in the production of sweat,
digestive juices and mucus.
• In CF individuals, mutations in the CTFR gene lead to thick mucous
secretions in the lungs and subsequent persistent bacterial
infections.
• The presence of CTFR mutations in a individual can be detected by
performing PCR and sequencing on that individual’s DNA.
Human Immunodeficiency Virus (HIV)
• HIV is a retrovirus that attacks the immune system.
• HIV tests rely on PCR with primers that will only amplify
a section of the viral DNA found in an infected
individual’s bodily fluids.
Therefore if there is a PCR product, the person is likely to be
HIV positive. If there is no PCR product the person is likely to
be HIV negative.
• Protein detection based tests are available as well but all US blood
is tested by PCR.
PCR and Forensic Science
• Forensic science is the application of a broad spectrum of sciences
to answer questions of interest to the legal system. This may be in
relation to a crime or to a civil action.
• It is often of interest in forensic science to identify individuals

genetically. In these cases, one is interested in looking at variable
regions of the genome as opposed to highly-conserved genes.
• PCR can be used to amplify highly variable regions of the human

genome. These regions contain runs of short, repeated sequences
(known as variable number of tandem repeat (VNTR) sequences) .
The number of repeats can vary from 4-40 in different individuals.
• Primers are chosen that will amplify these repeated areas and the
genomic fragments generated give us a unique “genetic fingerprint”
that can be used to identify an individual.
PCR Applications to Forensic Science
• Paternity suits -Argentina’s Mothers of the plaza

and their search for abducted grandchildren
• Identifying badly decomposed bodies or when only

body fragments are found - World trade center,
Bosnian , Iraq & Rwandan mass graves

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Polymerase Chain Reaction

• The most commonly used DNA polymerase is

• Mg2+ ions in the buffer act as co-factor for DNA

Similarly, if you subject your DNA of interest to 40 cycles of

• HIV is a retrovirus that attacks the immune system.

• It is often of interest in forensic science to identify individuals

• PCR can be used to amplify highly variable regions of the human

• Paternity suits -Argentina’s Mothers of the plaza

• Identifying badly decomposed bodies or when only

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