Details and Types of Polymerase Chain Reaction

2. Polymerase
technique
chain
used
reaction (PCR) is a
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.

3.  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.

4. DNA template is target DNA sequence. It is
the DNA molecule that contains the region
(segment) to be amplified, the segment we
are concerned which is the target sequence.

5. 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
a thermophillic bacterium)aquaticus,
because of high temperature stability.

6. DNA polymerasePfu (fromPyrococcus
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.

7. 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 .

8. Two primers must be designed for PCR; the
forward primer and the reverse primer.

9. The forward primer attaches to the start codon of the template DNA (the anti-
sense strand), while the reverse primer attaches to the stop codon of the
complementary strand of DNA (the sense strand).
The 5' ends of both primers bind to the 3' end of each DNA strand.

10. Primers should bind to template with good
specificity and strength. If primers do not
bind to correct template, wrong sequence
sequences and appropriate
will get amplified. Optimal primer
primer
concentrations are essential for maximal
specificity and efficiency in PCR.

11. Complementary nucleotide sequences within
a primer and between primers should be
avoided. If there are complimentary
sequences in two primers used (one primer
for each will
hybridize
DNA strand), the primers
with each other thus forming
primer-dimmers and will not be available for
binding with template. If there are
complementary sequences within a primer,
it will make hairpin loop structures as shown
below.

14.  The primers should preferably end on a
Guanine and Cytosine (GC) sequence so
that it can attach with sufficient strength
with template. This increases efficiency of
priming due to stronger bonding of G and C
bases.

15. All types of nucleotides are "building
blocks" fornew DNA strands and essential
for reaction. It includes Adenine(A),
Guanine(G), Cytosine(C), Thymine(T) or
Uracil(U).

16. Magnesium affects primer annealing and
template denaturation, as well as enzyme
activity.
An excess of magnesium gives non-specific
amplification products, while low magnesium
yields lesser amount of desired product.

17. 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.

18. • During the heating step (denaturation), the
reaction mixture is heated to 94°C for1min,
which causes separation of DNA double
stranded. Now, each strand acts as template for
synthesis of complimentary strand.

20. 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.

21. Guanine pairs
hydrogen bonding adenine binds
with cytosine with three
with
thymine with two hydrogen bonds. Thus,
higher GC content results in stronger
binding.

23. • 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.

25. First three steps are repeated 35-40 times to
produce millions of exact copies of the
target DNA. Once several cycles are
completed, during the hold step, 4–15 °C
temperature is maintained for short-term
storage of the amplified DNA sample.

26. • As both strands are copied during PCR, there is
an exponential increase of the number of copies
of the gene. Suppose there is only one copy of
the desired gene before the PCR starts, after one
cycle of PCR, there will be 2 copies, after two
cycles of PCR, there will be 4 copies. Afterthree
cycles there will be 8 copies and so on.

31. Polymerase Chain Reaction
1. Diagnosis of bacterial and viral diseases
In early phases of tuberculosis, the sputum may contain only
very few tubercle bacilli, so that usual acid fast staining may be
negative.
But PCR could detect even one bacillus present in the specimen. Any
other bacterial infection could also be detected.
The specific nucleotide sequences of the bacilli are amplified by PCR
and then detected by Southern blot analysis.

32. 2. Medicolegal cases
PCR allows the DNA in a single cell or in a hair follicle to be
analysed.
The restriction analysis of DNA from the hair follicle from the
crime scene is studied after PCR amplification.
This pattern is then compared with the restriction analysis of DNA
samples obtained from various suspects.
The culprit's sample will perfectly match with that of PCR
amplified sample.

33. 3. Diagnosis of genetic disorders
The PCR technology has been widely used to amplify the
gene segments that contain known mutations for diagnosis of
inherited diseases such as sickle cell anemia, thalassemia, cystic
fibrosis, etc.
4. Prenatal diagnosis of inherited diseases
5. Cancer detection: PCR is widely used to monitor residual
abnormal cells present in treated patients. Similarly, identification of
mutations in oncosuppressor genes such as p53, or retinoblastoma
gene can help to identify individuals at high risk of cancer.

35. Reverse Transcriptase PCR (RTPCR)
Instead of Taq polymerase, Tth polymerase from Thermus
Thermophilus may be used. This enzyme has both DNA
polymerase and reverse transcriptase activities at 95°C. So
mRNA is copied to cDNA synthesis followed by PCR
amplification. In ordinary PCR, DNA is detected; that
DNA could be from a living or non-living organism. But in
reverse PCR, mRNA is detected; that means, it is derived
from a living organism.

36. Reverse Transcriptase PCR (RTPCR)
By this method, quantitation of the number of virus
present in a sample can be calculated., e.g.,viral load in
HIV (human Immuno deficiency virus or HBV (hepatitis
B virus).

37. A real-time polymerase chain reaction (real-time PCR),
also known as quantitative polymerase chain
reaction (qPCR), is a laboratory technique of molecular
biology based on the polymerase chain reaction (PCR). It
monitors the amplification of a targeted DNA molecule
during the PCR (i.e., in real time), not at its end, as in
conventional PCR.

38. Real-time PCR is carried out in a thermal cycler with the capacity to detect the
fluorescence emitted by the dye applied.
The PCR process generally consists of a series of temperature changes that are
repeated 25–50 times.
These cycles normally consist of three stages:
the first, at around 95 °C, allows the separation of the nucleic acid's double
chain
the second, at a temperature of around 50–60 °C, allows the binding of the
primers with the DNA template
the third, at between 68–72 °C, facilitates the polymerization carried out by the
DNA polymerase.