PCR

SMIC601
Unit 2- Applications of rDNA

Technology & Bioinformatics
Unit 2.1- different types of PCR (Reverse transcriptase PCR, Real time
quantitative PCR)
Snehal Dabhade
Polymerase Chain Reaction ( PCR)
 Polymerase chain reaction (abbreviated
PCR) is a laboratory technique for
rapidly producing (amplifying) millions
to billions of copies of a specific
segment of DNA, which can then be
studied in greater detail.
 PCR involves using short synthetic DNA
fragments called primers to select a
segment of the genome to be
amplified, and then multiple rounds of
DNA synthesis to amplify that segment.
 From a simple basic principle, many
variations have been developed with
applications throughout gene
technology .
 Very importantly, the PCR has
revolutionized prenatal diagnosis by
allowing tests to be performed using
small samples of fetal tissue. In
forensic science, the enormous
sensitivity of PCR-based procedures is
exploited in DNA profiling.
Basic Reaction
 The basis of PCR is replication catalyzed by a DNA polymerase.
 Replication in this case has two essential requirements: (1) a single-stranded
DNA template from which a new DNA strand can be copied and (2) a pair of
primers with a 3′-OH group to which new nucleotides can be added.
 Because a DNA molecule consists of two nucleotide strands, each of which
can serve as a template to produce a new molecule of DNA, the amount of
DNA doubles with each replication event.
 The primers used in PCR to replicate the templates are short fragments of
DNA, typically from 17 to 25 nucleotides long, that are complementary to
known sequences on the template
The PCR Reaction
A solution that
includes the
target DNA, DNA
polymerase, all
four
deoxyribonucleosi
de triphosphates
(dNTPs—the
substrates for
DNA polymerase),
primers, and
magnesium ions
and other salts
that are
necessary for the
The PCR Reaction
 Step I: DNA is heated to between 90° and 100°C to break the hydrogen bonds between
the strands and thus produce the necessary single-stranded templates. The reaction
mixture is held at this temperature for only a minute or two.
 Step II: the DNA solution is cooled quickly to between 30° and 65°C and held at this
temperature for a minute or less. During this short interval, the DNA strands will not
have a chance to reanneal, but the primers will be able to attach to the template strands.
 Step III: the solution is heated for a minute or less to between 60° and 70°C, the
temperature at which DNA polymerase can synthesize new DNA strands. At the end of
the cycle, two new double-stranded DNA molecules are produced for each original
molecule of target DNA.
 The whole cycle is then repeated. With each cycle, the amount of target DNA doubles; so
the target DNA increases geometrically. One molecule of DNA increases to more than
1000 molecules in 10 PCR cycles, to more than 1 million molecules in 20 cycles, and to
more than 1 billion molecules in 30 cycles. Each cycle is completed within a few minutes;
so a large amplification of DNA can be achieved within a few hours.
Key Innovations Facilitating the PCR
 The first was the discovery of a DNA polymerase that is stable at the high
temperatures used in step 1 of PCR.
 The DNA polymerase from E. coli that was originally used in PCR denatures at 90°C.
For this reason, fresh enzyme had to be added to the reaction mixture in each
cycle, slowing the process considerably. This obstacle was overcome when DNA
polymerase was isolated from the bacterium Thermus aquaticus, which lives in the
boiling springs of Yellowstone National Park. This enzyme, dubbed Taq polymerase,
is remarkably stable at high temperatures and is not denatured in the strand-
separation step of PCR; so it can be added to the reaction mixture at the beginning
of the PCR process and will continue to function through many cycles.
 The second key innovation was the development of automated thermal cyclers—
machines that bring about the rapid temperature changes necessary for the
different steps of PCR. Originally, tubes containing reaction mixtures were moved
by hand among water baths set at the different temperatures required for the three
steps of each cycle. In automated thermal cyclers, the reaction tubes are placed in a
metal block that changes temperature rapidly according to a computer program.
Thermostable DNA polymerases that have proofreading activity are available for
Advantages & Limitations of PCR
PCR and such enzymes significantly decrease the error frequency. One such
enzyme is Vent polymerase, which was extracted from an archaean growing
around high-temperature deep-sea oceanic vents.
ADVANTAGES:
 PCR is a much more sensitive and quicker technique than cloning.
 Starting with just one molecule, PCR can produce millions of copies of a DNA
segment in just a few hours.
 By contrast, cloning requires a significant amount of starting DNA for
restriction digestion, and then at least a week is needed to go through all
the cloning steps
LIMITATION:
 PCR requires the use of specific primers
 the length of DNA that can be amplified by PCR is limited by the enzyme
and conditions to about 40 kb.
 the Taq polymerase used by many researchers has no proofreading activity.
RT PCR & mRNA Quantification
 PCR is used for some experiments in which the starting material is RNA. The
two examples described are reverse transcription-PCR, and real-time PCR.
 Reverse transcription-PCR (RT-PCR) is a method in which RNA first is
converted to cDNA and then the cDNA is amplified by PCR.
 RT-PCR is a very sensitive technique for detecting and quantifying RNA,
often mRNA.
 There are three steps to RT-PCR. First, cDNA is synthesized from RNA using a
primer (oligo(dT), for example, for mRNA) and the enzyme reverse
transcriptase (RT).
 Then the specific cDNA made is amplified by PCR using primers
complementary to the two ends of the molecule, and the PCR products are
Oligo(dT) is a short segment of deoxythymidine nucleotides(12-18)
analyzed using gel electrophoresis.
that is used as a primer in cDNA synthesis. It initiates the
synthesis of the first strand of cDNA by annealing to the 3' end of
poly(A)
Random Primers
These are the short single-stranded sequences of 6 to 8 nucleotides that bind at the
complementary site of RNAs with or without poly(A) for cDNA synthesis using reverse
transcriptase.
Sequence-specific Primers
These are the short single-stranded sequences of nucleotides that bind to the
Advantages & Applications of RT- PCT
ADVANTAGES:
 The RT-PCR technique,like regular PCR, is a very sensitive technique, in that
it will be able to tell us that our mRNA is present, even if our mRNA makes
up only a tiny fraction of the starting RNA pool.
APPLICATIONS:
 RT-PCR is used either for testing for the presence of a particular RNA, or for
roughly quantifying the amount of an RNA.
 some viruses have RNA genomes and theoretically RT-PCR could be used to
detect whether an individual has been infected by the virus. Such tests have
been developed for HIV, measles virus, and mumps virus.
LIMITATION:
 it is generally impossible to determine exact abundance of the mRNA for a
gene of interest
 https://wordwall.net/resource/82751190

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SMIC601

Unit 2- Applications of rDNA

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