Types of PCR
Allele-specific PCR is a varation of the polymerase chain reaction which is used as a diagnostic or cloning technique, to identify or utilize single-nucleotide polymorphisms (SNPs) (single base differences in DNA). Allele-specific PCR does require the sequence of the target DNA sequence, including differences between the alleles.
Primers for Allele-Specific PCR
The allele-specific PCR uses primers whose 3' ends encompass the SNP.
PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with a SNP-specific primer signals presence of the specific SNP in a sequence. Single nucleotide polymorphism (SNP, pronounced snip), is a DNA sequence variation occurring when a single nucleotide - A, T, C, or G - in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual). For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case we say that there are two alleles : C and T. Almost all common SNPs have only two alleles. For a variation to be considered a SNP, it must occur in at least 1% of the population.
Assembly PCR is a polymerase chain reaction variation that artificial synthesizes long DNA sequences by performing PCR on a pool of long oligonucleotides (primers) with short overlapping segments.
The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product.
Asymmetric PCR is used to preferentially amplify one strand of the target DNA more than the other.The Asymmetric PCR is useful in some sequencing and hybridization probing applications where having only one of the two complementary stands is sufficient or required.
The asymmetric PCR method is conducted as the standard pcr protocol, however a great excess of the primers for the chosen strand is used.
Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.
Primers for Asymmetric PCR
A PCR in which the predominant product is a single-stranded DNA, as a result of unequal primer concentrations.
As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA. The higher concentration primer continues to primer synthesis, but only of its strand.
A recent modification of assymetric PCR is Linear-After-The-Exponential-PCR or LATE-PCR.
Primers for LATE-PCR
LATE-PCR utilizes 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.
Colony PCR the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products. Selected colonies of bacteria or yeast are picked with a sterile toothpick or pipette tip from a growth (agarose) plate. This is then inserted into the PCR master mix or pre-inserted into autoclaved water. PCR is then conducted to determine if the colony contains the DNA fragment or plasmid of interest.
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