Lectut btn-202-ppt-l27. variants of pcr-i
- 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 2
- 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. 3
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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. 5
- 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 6
- 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. 7
- 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. 8
- 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. 9
- 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. 10
- 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. 11
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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. 13
- 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. 14
- 15. Applications of IS-PCR In situ PCR amplification can be performed on fixed tissue or cells or on a slide . 15
- 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 16
- 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. 17
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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. 19
- 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. 20
- 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. 21