CO amplification at Lower Denaturation temperature PCR (#COLDPCR) is a variant of #PCR aimed at enriching mutated #DNA sequences to be ultimately analyzed by sequencing. Just by adding a step in the conventional PCR cycle, this can be achieved utilizing the concept of #CriticalDenaturationTemperature (Tc).
FIGURE: Working principle of COLD-PCR.
Principle:
The principle of COLD PCR entirely depends on the concept of critical denaturation temperature. Every stretch of dsDNA has a critical denaturation temperature (Tc) that is lower than its #meltingtemperature (Tm) which depends on its base constitution obviously. Even a single or double nucleotide mismatches between two different #amplicons can lead to very different amplification efficiencies if the PCR is set at Tc in the denaturation step. Any temperature below this Tc drastically pulls down PCR amplification efficiency for that target #dsDNA stretch.
in COLD PCR, the mismatch containing dsDNA stretches get amplified because the denaturation temperature is set at Tc specific for the mutant allele in the interrogated DNA sequence.
Protocol:
Tc determination: The Tc of the mutated DNA sequence is ascertained by generally setting up #RealTimePCR and subsequent melt curve analysis to get the melting point (Tm). The Tc for full- and fast-COLD-PCR is typically 1 C below the experimentally-derived amplicon melting temperatures (Tm). Defining the Tc in this empirical manner (Tc=Tm-1) results to robust PCR amplification as well as substantial mutation enrichment.
Primer designing: There are also no special rules for primer designing here, although it is suggested that Ta of the primers should be less than 65 C to prevent interference with heteroduplex formation.
full COLD PCR
a. Denaturation: After some cycles (8-10 PCR cycles) of general PCR, the amplicons are heated to 94-98C for denaturation.
b. Intermediate annealing: The PCR amplicons are allowed to cross-hybridize at an intermediate temperature (~70 C for 2–8 min). As mutant alleles are less in numbers, most mutant alleles end up in a mismatch-containing structure (‘#heteroduplex’) that has a lower Tm than the fully matched structure (‘#homoduplex’).
c. Heteroduplex denaturation: The temperature is raised to the Tc (~80-90 C), to denature the heteroduplexes preferentially over the fully matched sequences. Because critical denaturation is performed at every PCR cycle, the differential enrichment of mutation-containing alleles is compounded exponentially, and results in a large difference in overall amplification efficiency between mutant and wild-type alleles, at the end of the cycling.
d. Primer annealing: The temperature is reduced to 55 C to allow primers to bind and replicate the preferentially denatured sequences.
e. Extension: The temperature is raised to 72 C for the Taq to extend the 3' end of the bound primers and the cycle continues upto your patience!
Sub-types:
1. full COLD PCR
2. fast COLD PCR
3. Temperature-tolerant COLD PCR
4. fast temperature-gradient COLD PCR
5. Improved and Complete Enrichment COLD-PCR (ice COLD PCR)
6. Enhanced ice COLD PCR
Merits:
1. It can preferentially amplify minority alleles even in the presence of excess of the wild-type variant.
2. It can enrich low-level somatic DNA mutations from a mixture of input DNA sample.
3. Mutation detection sensitivities are enhanced manifolds by combining COLD-PCR with #Sanger sequencing, #dHPLC analysis, #MALDI-TOF, Pyrosequencing, real-time #TaqMan PCR, #SSCP, mutation-specific restriction enzyme digestion and high resolution melting.
4. No additional reagents or cumbersome PCR setups are needed, relatively easy to grasp.
Demerits:
1. Optimal Tc must be ascertained for each amplicon beforehand, adding a lot of tedious and repetitive work.
2. It requires precise denaturation temperature control during PCR to the tune of ~0.3 C.
3. A suitable Tc might not be available for all possible amplicons, adding to its limitation.
4. It is restricted to amplicons smaller than or equal to 200bp.
5. No certainty of all minor alleles being enriched.
6. Overall mutant DNA enrichment depends on primer positions and base substitution.
Now get back to the bench!
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