Who all of you can't miss ice on your drinks? well ice does make things better, i mean atleast here! There have been advantages and disadvantages of #fastCOLDPCR and #fullCOLDPCR as we have already discussed. So to surpass the disadvantages and combine the advantages of both, the Improved and Complete Enrichment COLD PCR (#iceCOLDPCR)came into being.
Principle
It keeps intact the basic principle of Critical Denaturation Temperature (#Tc) as per any COLD PCR. The modification comes with a #ReferenceSequence (RS) which is basically an #HPLC-purified oligonucleotide almost of the length of the amplicon. The RS has some characteristics as:
a. It is #complementary to the wild type sense strand sequence.
b. It has <= 5bp overlap with amplicon primers to prevent primer binding to itself.
c. It's 3' PO4 prevents polymerase extension, providing a second layer of protection against non-specific amplification
d. The RS is used in excess in the PCR reaction to the tune of ~25nM.
When in the PCR reactions in excess amounts relative to the template, the RS binds rapidly to the #amplicons after denaturation. At Tc, the RS:WT(wild-type) duplexes remain double-stranded, thereby inhibiting selectively the amplification of WT alleles throughout the #thermocycling. Conversely, the RS:mutant duplexes are preferentially denatured and amplified. By using a WT-specific RS, all #variants can be effectively amplified, regardless of mutational type and position. Using an #oligonucleotide synthesizer to construct the RS is convenient but it has oligonucleotide-length limitations. Alternatively, longer RS can be synthesized using #asymmetricPCR approaches, followed by end-modification.
Protocol
The protocol is a subtle modification of the full-COLD PCR protocol.
FIGURE: ice-COLD PCR general #cyclingconditions.
FIGURE: Schematics of mutant enrichment in ice-COLD PCR.
The qPCR reaction mix contains 5X Phusion HF (high fidelity) buffer, 1.5mM #MgCl2, 0.2mM #dNTPs, 0.3 uM primers, 0.1X #LCGreen+ dye (#IdahoTechnologies), 5U/ul #PhusionTM high fidelity polymerase (#Finnzymes), 25nM RS oligonucleotide and template DNA.
Merits:
1.ice-COLD PCR results in up to a 500-fold increase in sensitivity in identifying mutations compared to conventional PCR methods.
2. The inclusion of 60-90 nt long RS within COLD PCR reactions selectively enhances denaturation of mutant over WT sequences throughout PCR, while also reducing time-intensive #hybridization stages as in full-COLD PCR.
3. Inclusion of a non-amplifying WT RS sequence in ice-COLD PCR increases the mutational abundance >50% (relative to the WT allele); per contra enrichment >50% cannot be achieved using full- COLD-PCR.
4. In addition to Tm-reducing mutations, low-abundance Tm increasing and Tm-equivalent mutations can also be enriched easily, robustly and rapidly via ice-COLD PCR, and then subsequently identified through #sequencing.
5. While #Sangersequencing of ice-COLD PCR amplicons demonstrated the ability to identify a 1% mutant, #pyrosequencing allows the user to identify a 0.1% mutant mixture, demonstrating a 10-fold advantage in sensitivity based upon downstream sequencing-methodology.
Give it a shot!
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