Wednesday, August 17, 2011

Variations of the basic PCR techniques

Multiplex-PCR: In this test, multiple unique primer sets are used within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run saving the reagents and time. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction and the base pair length amlicons should be different enough to form distinct bands when visualized by gel electrophoresis ( Hayden et al.,2008).

Nested PCR : Is a modification that can be used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first one : pair of primers are used to generate DNA products which may also contain products amplified from non-target areas. The products from the first PCR are then used to start the second reaction

using one (hemi-nesting) or two different primers whose binding sites are located (nested) within the first set. Nested PCR is often more successful in specifically amplifying long DNA products than conventional PCR but it requires more detailed knowledge of the sequence of the target ( Pierce , Wangh, 2007 & Park 2004).

Quantitative PCR (Q-PCR):The example of this modification is real-time PCR. It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine the presence of a DNA sequence in a sample and the number of its copies. In this method , fluorescent dyes such as Sybr Green or fluorophore-containing DNA probes such as TaqMan are used to measure the amount of amplified product in real time ( Pavlov et al ., 2006).

Hot-start PCR : Is a technique that modifies the way that a PCR mixture is initially heated. During this step the polymerase is active but the target has not yet been denaturated and the primers may be able to bind to non-specific locations (or even to each other). The technique can be performed manually by heating the reaction components to the melting temperature (as 95°C) before adding the polymerase. Alternatively specialized systems have been developed that inhibit the polymerase's activity at certain temperature either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step.Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are only activated at elevated temperatures (Saiki et al., 1988).

Touchdown PCR : In this technique , the temperature used

to anneal the primers is gradually decreased in later cycles. The annealing temperature in the early cycles is usually 3-5°C above the standard Tm of the primers used while in the later
cycles it is 3-5°C below the Tm. The initial higher annealing temperature leads to greater specificity for primer binding while the lower temperatures permit more efficient amplification to the end of the reaction ( Don et al ., 1991).

Assembly PCR : (also known as Polymerase Cycling Assembly or PCA) is the artificial synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotide building blocks alternate between sense and antisense directions, and the overlaps determine the order of oligonucleotides, selectively producing the final long DNA product (Myriam et al ., 2004).

RT-PCR : (or Reverse Transcription PCR) is a common method used to amplify, isolate or identify a known sequence from a cell's or tissue's RNA. PCR is preceded by a reaction using reverse transcriptase which converts RNA into cDNA. Amplification of cDNA is then usually performed by a thermostable and thermoactive DNA Taq polymerase. Another DNA polymerase has been recently isolated from thermous thermophilus (rTh polymerase) (Myers & Gelfand, 1991) and is used for coupled reverse transcription (RT) and PCR. The reaction conditions for both RT & DNA amplification occur in a single manganese buffer (Tang & Persing , 1999).
The two reactions are compatible enough that they can be run in the same mixture tube with the initial heating step of PCR being used to inactivate the transcriptase. RT-PCR is widely used in expression profiling which determines the expression of a gene or identifies the sequence of an RNA transcript

including transcription start and termination sites (Pierce & Wangh 2007).

Methylation-specific PCR: (or MSP) is developed to study patterns of methylation in genomic DNA. Target DNA is first treated with sodium bisulfate which converts unmethylated cytosine bases to uracil which is recognized by PCR primers as thymine. Two amplifications are then carried out on the modified DNA using primer sets that distinguish between the modified and unmodified templates. One primer set recognizes DNA with cytosine to amplify the previously methylated DNA and the other set recognizes DNA with uracil or thymine to amplify unmethylated target. MSP using Q-PCR can also be performed to obtain quantitative information about methylation (Park , 2005).

Asymmetric PCR: Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary strands is required. PCR is carried out as usual but with a great excess of the primers for the choosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used , extra cycles of PCR are required. A recent modification of this process is known as Linear-After-The-Exponential-PCR (LATE-PCR) uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction ( Isenbarger et al ., 2008).

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