Biotechnology Engineering

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Lecture- 27

2. VARIANTS OF PCR
 Inverse PCR
 Colony PCR
 Hot Start PCR
 Multiplex PCR
 In situ PCR (IS-PCR)
 Long PCR
 Nested PCR
 Touchdown PCR
 Reverse transcriptase-PCR(RT-PCR)
 Band-stab PCR
 Degenerate PCR
 Anchored PCR
 Ligated –anchored PCR(LA-PCR)
 Asymmetric PCR
 Differential Display Reverse Transcriptase PCR (DDRT-PCR)
 Real-time PCR
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3. INVERSE PCR
 Inverse PCR is used to amplify unknown DNA that flanks one end of
known DNA sequence for which no primers are available .
 In this PCR information of only one internal sequence of the target DNA is
required. So, It is very useful in identifying flanking DNA sequences of
genomic inserts.
 This technique involves digestion by restriction enzyme of a DNA
preparation having known DNA sequence and its flanking sequence. The
restriction fragments are self-ligated and changed into circular DNA and the
circularized DNA is used as a template in the PCR reaction.
 The primers are designed from known sequence that faces outward from
that region .The resulting amplified product will be a single linear fragment
that includes unknown DNA from both left and right sides. The PCR
product can be cloned and then sequenced.
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5. Inverse PCR Applications
 Amplification and identification of sequences flanking
transposable elements
 Identification of genomic inserts
 Cloning of unknown cDNA sequences from total RNA
 Construction of end-specific probes for chromosome walking
 Amplification of integration sites used by viruses and
transgenes.
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6. COLONY PCR
 Colony PCR is used for screening recombinants from
bacterial, bacteriophage or yeast transformation products.
 A colony is picked with a sterile toothpick or pipette tip
from a growth plate.
 This is then inserted into autoclaved water and PCR is
then conducted
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7. Applications of colony PCR
 Colony PCR is a fast and reliable method for the
screening of recombinants.
 A number of colonies or plaques can be assayed
simultaneously and there is no need to store a large
number of transformed clones for long periods.
 This method can easily be used for cDNA library
screening.
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8. HOT START PCR
 Hot Start PCR permits the inhibition of polymerase activity during
setting of PCR reaction, by limiting polymerase activity prior to PCR
cycling.
 The primers, Mg2++, buffer and dNTPs can be mixed at room
temperature in the bottom of the PCR tube and then covered with
melted wax (e.g., Ampliwax PCR Gems from Perkin-ELMER). The
wax solidifies on cooling and limits the reagents to bottom of the
tube.
 The remaining components are then added on top of the barrier. Wax
layer melts upon heating during the denaturation step and the two
aqueous layers get mixed resulting in a fully active reaction.
 The melted wax floats to the top of the reaction mixture where it acts
as a barrier to evaporation.
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9. Automated Hot Start PCR
 More recently, an alternate method of automated Hot Start PCR
has been developed where Taq DNA polymerase-directed
monoclonal antibodies, as thermolabile inhibitors of enzymes,
are used.
 At low temperature, i.e., at ambient temperature antigen-
antibody interaction results in potent inhibition of DNA
polymerase;
 When temperature rises at the start of thermocycling process,
the antibodies are denatured and fully active Taq DNA
polymerase is released.
 The pre-incubation of Taq DNA polymerase and antibodies
results in highly specific PCR reaction with increased
sensitivity.
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10. Applications of Hot Start PCR
 Hot Start PCR reduces nonspecific amplification and
increases the sensitivity, specificity, precision of
amplification of low copies of target DNA and yield of
PCR product.
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11. MULTIPLEX PCR
 All sequences of interest are simultaneously amplified in
multiplex PCR.
 Multiplex PCR has significant template, time and cost
saving advantages, especially when a large number of
individual sequences need to be analyzed. Single aliquot
of DNA or RNA is required rather than an aliquot for each
marker to be analyzed.
 Generally up to eight primer pairs can be used in a
standard multiplex reaction, otherwise the yield of some
amplicons is reduced and not visible on agarose gel.
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13. Applications of Multiplex PCR
 This type of PCRs is important for forensic application,
prenatal diagnosis, and for clinical applications in which
the tissue/DNA samples are limited.
 It is also used for genotyping applications where
simultaneous analysis of multiple markers is required.
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14. In situ PCR (IS-PCR)
 It is primer driven amplification of a DNA or RNA
template by PCR and its subsequent detection within the
histological tissue section or cell preparation.
 Preparation of sample for IS-PCR, i.e., fixation and
permeabilization
 Actual PCR
 Detection of amplified signal
 It is somewhat difficult to detect the genes of low copy
number by in situ PCR as it is below the detection limit.
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15. Applications of IS-PCR
 In situ PCR amplification can be performed on fixed
tissue or cells or on a slide .
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16. Long PCR
 Long PCR is a PCR, which is extended longer than standard PCR,
over 5 kbp (frequently. over 10 kbp).
 In case of standard PCR it is very difficult to get long PCR product
because of damage to the template and product DNAs due to
exposure to high temperature, presence of Mn2+ and difficulties in
denaturing of long DNA molecules.
 There are recent reports of amplification of 42 kbp with the blend of
enzymes primarily containing non-proofreading polymerase with a
very small amount of proofreading polmerase.
(i)45:1 Able to amplify a 22 kbp fragment of ß globin
from genomic DNA
(ii)125:1 Able to amplify 39 kbp of λ DNA
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17. Nested PCR
 In case of nested PCR two or more pairs instead of one
pair of PCR primers are used to amplify a fragment.
 The first pair of PCR primers amplifies a fragment similar
to a standard PCR.
 Internal primers, called nested primers as these hybridize
to the sites nested within the first fragment, are used to
amplify PCR products formed by external primers.
 The binding of nested primers inside the first PCR product
fragment allows amplification of a second PCR product
that is shorter than the first one.
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19. Advantages of Nested PCR
 The advantage of nested PCR is that if the wrong PCR
fragment is amplified, the probability is quite low that the
region is amplified a second time by the second set of
primers.
 Thus, nested PCR is used to increase magnitude and
specificity of amplification.
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20. Touchdown PCR
 In touchdown PCR, the Ta, during the first two cycles is
set at ~3oC above the calculated Ta.
 The annealing temperature is then reduced by one degree
centigrade for every one or two cycles.
 When the Ta, reaches at an optimum point, then specific
amplification of the target DNA starts.
 The onset of nonspecific amplification is delayed for few
additional cycles until the Ta, is lowered to the point where
nonspecific amplification priming can occur.
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21. Applications of Touchdown PCR
 It is used to optimize yield of amplified product at
different annealing temperatures.
 In some cases it is very tricky to amplify DNA because of
mismatches between oligonucleotide primers and the
template DNA and it is not possible to calculate annealing
temperature.
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