Variants of PCR 1. Gradient PCR 2. Touch Down PCR 3. Multiplex PCR 4. Asymmetric PCR 5. Allele Specific PCR 6. Colony PCR 7. Nested PCR 8. Hot Start PCR 9. RT-PCR 10. qPCR

1. Variants of PCR
PRESENTED BY: AYESHA KABEER
UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES,
LAHORE

2. 1. Gradient
PCR
Variant of conventional PCR
which facilitates the optimization
of PCR reaction by determining
the exact annealing temperature.
Best annealing temperature for
PCR is 60°C as shown.

3. 2. Touch
Down PCR
Variant of conventional PCR in
which the high specificity of
amplification is achieved by
reducing the unwanted
amplification on sequentially
decreasing the annealing
temperature after each PCR
cycle.
In simple words, Touch Down
PCR is a method to decrease off-
target priming and hence to
increase the specificity of PCRs.

4. 3. Multiplex
PCR
Variant of PCR in which more
than one target sequence is
amplified using multiple sets of
primers within a single PCR
mixture.
This enables amplification of
several gene segments at the
same time, instead of specific test
runs for each.

5. 4. Asymmetric
PCR
Variant of PCR which
preferentially amplifies one DNA
strand in a double-stranded
DNA template.
Thus it is useful when
amplification of only one of the
two complementary strands is
needed such as in sequencing
and hybridization probing.

6. 5. Allele Specific
PCR
Variant of PCR that permits the direct
detection of any point mutation or single
nucleotide polymorphism in human DNA by
analyzing the PCR products in an ethidium
bromide-stained agarose or polyacrylamide
gel.

7. 6. Colony
PCR
Colony PCR is a rapid, high
throughput PCR method to
determine the presence or
absence of the inserted DNA into
plasmid directly from the
bacterial colonies.
Primers designed to specifically
target the insert DNA can be
used to determine either the
construct contains the DNA
fragment of interest or not.

8. 7. Nested PCR
Variant of PCR which increases the specificity
of DNA amplification by reducing the non-
specific amplification of DNA using two
primer sets directed against the same target
and two successive PCR reactions.

9. 8. Hot Start PCR
Variant of PCR which reduces the non-
specific bindings by limiting one of the
reagents until the heating step of the PCR.

10. 9. RT PCR (Reverse
Transcriptase PCR)
Variant of PCR that allows genes to be
amplified and cloned as intron-free DNA
copies by starting with mRNA and using
reverse transcriptase.

11. 10. qPCR
(Quantitative PCR or
Real-Time PCR)
Variant of standard PCR in which
amplification and simultaneous quantitation
of a target DNA is done in the same PCR
machine, using commercially available
fluorescence-detecting thermocyclers.