What is PCR ? Steps in PCR Applications of PCR

1. POLYMERASE CHAIN REACTION
(PCR)
Shreyaskumar Jitendra Patel

2. PCR
• A technique used in molecular biology to amplify a single copy or a few copies of a
desired segment of DNA across several orders of magnitude, generating thousands to
millions of copies of a particular DNA sequence.
• It is an easy, cheap, and reliable way to repeatedly replicate a focused segment of
DNA, a concept which is applicable to numerous fields in modern biology and related
sciences.
• It was developed in 1983 by Kary Mullis and is now a common and often
indispensable technique used in clinical and research laboratories for a broad variety of
applications.
• PCR actually amplifies only a chosen segment (target sequence) within the original
DNA template, not the whole template DNA molecule.

3. Components used in PCR
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that is to be copied is
called the template,
the segment of it that
will actually be
amplified is known as
the target sequence.
original DNA molecule
needed to initiate DNA
synthesis. These are
short pieces of single
stranded DNA that
match the sequences
at either end of the
target DNA segment
Two PCR primers
needed to manufacture the
DNA copies. The PCR
procedure involves several
high temperature steps so a
heat resistant DNA
polymerase is required.
enzyme DNA polymerase
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Thermostable polymerases are isolated from bacteria living in hot springs at temperatures
up to 900 C. Taq polymerase from Thermus aquaticus is most widely used as it is stable at
high temperatures remaining active even after DNA denaturation, thus obviating the need
to add new DNA polymerase after each cycle. This allowed an automated thermocycler-
based process for DNA amplification.

4. Components used in PCR
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A supply of nucleotides
is needed by the
polymerase to make
the new DNA. These
are supplied as the
nucleotide
triphosphates/deoxynu
cleotide triphosphates
(dNTPs).
nucleotides
needed to keep changing the
temperature.
The PCR process requires cycling
through several different
temperatures. Because of this,
PCR machines are sometimes
called thermocyclers.
The reaction is commonly carried
out in a volume of 10–200 μl in
small reaction tubes (0.2–0.5 ml
volumes) in a thermal cycler.
PCR machine
providing a suitable
chemical environment
for optimum activity
and stability of the
DNA polymerase.
buffer solution
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5. Components of
PCR

6. CYCLING THROUGH PCR
● Typically, PCR consists of a series of 20–40 repeated
temperature changes, called cycles.
● There are 3 basic steps in each cycle of PCR:
template denaturation,
primer annealing
elongation of new strands of DNA.

7. Steps in PCR Cycle
This step is only required for DNA
polymerases that require heat
activation by hot-start PCR. It
consists of heating the reaction
chamber to a temperature of 94–96
°C, or 98 °C if extremely
thermostable polymerases are
used, which is then held for 1–10
minutes.
1. Initialization :
If initialization is not required, the template
DNA is denatured by heating to 90 ° C for
a minute. This causes DNA melting or
denaturation of the double stranded DNA
template by breaking the hydrogen bonds
between complementary bases, yielding
two single stranded DNA molecules.
Although the primers are present from the
beginning, they cannot bind to the template
DNA at 90 ° C
2. Denaturation :

8. Steps in PCR Cycle
The temperature is dropped to around 50-60 ° C for 20–40 seconds allowing the primers to
anneal to complementary sequences on the template strand. A longer primer is more
specific for binding to the exact target sequence.
Two different primers are typically included in the reaction mixture: one for each of the two
single-stranded complements containing the target region. The primers are single-stranded
sequences themselves, but are much shorter than the length of the target region,
complementing only very short sequences at the 3' end of each strand.
It is critical to determine a proper temperature for the annealing step because efficiency and
specificity are strongly affected by the annealing temperature. This temperature must be low
enough to allow for hybridization of the primer to the strand, but high enough for the
hybridization to be specific, i.e., the primer should bind only to a perfectly complementary
part of the strand, and nowhere else.
3. Annealing :

9. Steps in PCR Cycle
The temperature is increased to 700C for a minute or two to allow the thermostable
polymerase to elongate new complementary DNA strands starting from the primers. DNA
synthesis goes from 5’ to 3’ for both new strands. This gives 2 partly double stranded pieces
of DNA. The 2 new strands are not as long as the original templates. They are each missing a
piece at the end where synthesis started.
However, they are double stranded over the region that matters, the target sequence. The 3
steps are repeated in each cycle of PCR.
After repeating the same 3 steps, the second cycle produces 4 partly double stranded pieces
of DNA. Although they vary in length, they all include double stranded DNA from the target
region.
As the cycles continue, the single strand overhangs are rapidly outnumbered by segments of
DNA containing only the target sequence. Once past the first two or three cycles, the vast
majority of the product is double-stranded target sequence with flush ends.
4. Extension :

10. Steps in PCR Cycle
This single step is optional, but is
performed at a temperature of 70–
74°C (the temperature range
required for optimal activity of most
polymerases used in PCR) for 5–
15 minutes after the last PCR cycle
to ensure that any remaining single
stranded DNA is fully elongated.
5. Final elongation :
The final step cools the reaction chamber
to 4–15 °C for an indefinite time, and may
be employed for short-term storage of the
PCR products.
6. Final hold :
Finally, the DNA generated is run on an agarose gel
to assess the size of the PCR fragment.

12. ADVANTAGES
PCR has a number of advantages. It is fairly simple
to understand and to use, and produces results
rapidly.
The technique is highly sensitive with the potential
to produce millions to billions of copies of a specific
product for sequencing, cloning, and analysis.
qRT-PCR shares the same advantages as the
PCR, with an added advantage of quantification of
the synthesized product.
Therefore, it has its uses to analyze alterations of
gene expression levels in tumors, microbes, or
other disease states
DISADVANTAGES
One major limitation of PCR is that prior information
about the target sequence is necessary in order to
generate the primers that will allow its selective
amplification.
This means that, typically, PCR users must know
the precise sequence(s) upstream of the target
region on each of the two single stranded
templates in order to ensure that the DNA
polymerase properly binds to the primer-template
hybrids and subsequently generates the entire
target region during DNA synthesis.
Like all enzymes, DNA polymerases are also prone
to error, which in turn causes mutations in the PCR
fragments that are generated.

13. A researcher is performing PCR to
amplify a sample of DNA.
Unfortunately, he forgot to add the
DNA primer prior to starting the
experiment. Which of the following
results is he most likely to
observe?
A. The reaction will work, but at a
significantly slower rate
B. The reaction will work, but the product
will contain many undesired mutations
C. The reaction will be completely
unsuccessful
D. The reaction will work, but amplify a
region that was not his target
After four cycles of
thermocycling, how many
copies of the targeted
region will be in the PCR
product?
A. 2 B. 32
C. 64 D. 16

14. Majority of the time PCR amplifications
include an Initialization step, also
known as the Hot Start. What is the
purpose of the Hot Start?
A. Denaturation of the DNA strands
B. Activation of the DNA polymerase
C. Extension of the template strand
D. For primers to begin binding to the template strand
Adenylation of the new DNA strands by DNA

15. Thank You