Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR)
PRESENTED BY
( IMRAN ALIZAI)
PCR
Defination
T he polymerase chain reaction (PCR) is a
technique to amplify a single or few copies of
a piece of DNA across several orders of
magnitude, generating thousands to millions
of copies of a particular DNA sequence.
BASIC REAGENTS REQUIRES FOR PCR
• DNA template that contains the DNA region (target) to be

amplified.
• Two primers that are complementary to the 3' (three prime)
ends of each of the sense and anti-sense strand of the DNA
target.
• Taq polymerase or another DNA polymerase with a
temperature optimum at around 70 °C.
• Deoxynucleoside triphosphates (dNTPs; also very commonly
and erroneously called deoxynucleotide triphosphates), the
building blocks from which the DNA polymerases synthesizes
a new DNA strand.
• Buffer solution, providing a suitable chemical environment for
optimum activity and stability of the DNA polymerase.
PROCEDURE
• The PCR usually consists of a series of 20-40
repeated temperature changes called cycles;
each cycle typically consists of 2-3 discrete
temperature steps. Most commonly PCR is
carried out with cycles that have three
temperature steps
Different Stages of PCR
• 1. Initialization step
• 2. Denaturation step
• 3. Annealing step
• 4. Extension step
• 5. Final elongation Step
Initialization step:
• This step consists of heating the reaction to a
temperature of 94–96 °C (or 98 °C if extremely
thermostable polymerases are used), which is
held for 1–9 minutes. It is only required for
DNA polymerases that require heat
activation .
Denaturation step:
• This step is the first regular cycling event and
consists of heating the reaction to 94–98 °C
for 20–30 seconds. It causes DNA melting of
the DNA template by disrupting the hydrogen
bonds between complementary bases,
yielding single strands of DNA.
Digramic view of
Denaturation step
PCR Primers
Primers range from 15 to 30 nucleotides, are
single-stranded, and are used for the
complementary building blocks of the target
sequence
PCR Primers
A primer for each target sequence on the end
of your DNA is needed. This allows both
strands to be copied simultaneously in both
directions.
PCR Primers
TTAACGGCCTTAA . . . TTTAAACCGGTT
AATTGCCGGAATT . . . . . . . . . .>
and
<. . . . . . . . . . AAATTTGGCCAA
TTAACGGCCTTAA . . . TTTAAACCGGTT
PCR Primers
The primers are added in excess so they will
bind to the target DNA instead of the two
strands binding back to each other.
Annealing step:
• The reaction temperature is lowered to 50–
65 °C for 20–40 seconds allowing annealing of
the primers to the single-stranded DNA
template. Typically the annealing temperature
is about 3-5 degrees Celsius below the Tm of
the primers used. Stable DNA-DNA hydrogen
bonds are only formed when the primer
sequence very closely matches the template
sequence. The polymerase binds to the
primer-template hybrid and begins DNA
synthesis.
Digramic view of
Annealing step
Extension step:
• The temperature at this step depends on the DNA
polymerase used; Taq polymerase has its optimum
activity temperature at 75–80 °C, and commonly a
temperature of 72 °C is used with this enzyme. At
this step the DNA polymerase synthesizes a new DNA
strand complementary to the DNA template strand
by adding dNTPs that are complementary to the
template in 5' to 3' direction, condensing the 5'-
phosphate group of the dNTPs with the 3'-hydroxyl
group at the end of the nascent (extending) DNA
strand.
Digramic view of
Extension step
Final elongation Step:
• This single step is occasionally performed at a
temperature of 70–74 °C for 5–15 minutes
after the last PCR cycle to ensure that any
remaining single-stranded DNA is fully
extended.
Digramic view of
Final elongation Step:
Applications of PCR
Because PCR amplifies the regions of DNA that it targets, PCR
can be used to analyze extremely small amounts of sample.
This is often critical for forensic analysis, when only a trace
amount of DNA is available as evidence. PCR may also be used
in the analysis of ancient DNA that is tens of thousands of
years old. These PCR-based techniques have been successfully
used on animals, such as a forty-thousand-year-old
mammoth, and also on human DNA,
Quantitative PCR methods allow the estimation of the
amount of a given sequence present in a sample – a
technique often applied to quantitatively determine levels of
gene expression. Real-time PCR is an established tool for DNA
quantification that measures the accumulation of DNA
product after each round of PCR amplification.
Thank You

Polymerase Chain Reaction (PCR)

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Polymerase Chain Reaction (PCR)

• DNA template that contains the DNA region (target) to be

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