Variants of PCR

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.