This 'COP' detects #pointmutations with flair! The #CompetitiveOligonucleotidePrimingPCR or #copPCR is one of the simplest modification of #conventionalPCR to enrich mutations present in a mixed pool with #wildtypealleles.
Principle
FIGURE: The general principle of #copPCR.
The basic principle of #copPCR is a favored #amplification of the #templateDNA in presence of a two competing #oligonucleotides with one of them matching the template perfectly. Both the primers are mixed in a single annealing reaction with template with each of them capable of priming DNA synthesis at the same site. When one of them is perfectly complementary to the template it can bind in preference to the one that differs by a single base. The use of a third common primer allows the identification of the successfully competing primer by #PCR.
When the primer-annealing reactions are carried out at low stringency and with an excess primer to template ratio, the perfectly matched primer can be favored with a level of discrimination greater than 100:1. The perfectly matched primer can also compete successfully when it is at #LowAbundance (i. e., in the presence of up to a 100-fold molar excess of a mismatched primer) or when the correct match is only one of a four member mixed #oligonucleotide family.
The detection can be achieved in two different ways:
a. Differential labeling of primers: This followed by application to anti-label #LateralFlowStrips would provide a reliable and easy detection of #PointMutations.
b. Labeled and unlabeled primers: This followed by #DeferentialMigration of the PCR products owing to the label, can provide a detection alternative.
Protocol
The protocol is same as the #ConventionalPCR with following modifications, keeping cycling conditions conventional:
a. There is one common reverse primer along with two competing forward primers used.
b. The #MismatchPrimer has to have the mutant base at the middle of its sequence.
c. The primers has to be shorter than used in #ConventionalPCR, ideally 12-mers to 16-mers.
d. The primer concentration used is more than used in #ConventionalPCR, generally 0.5-2 uM (0.5-2 picomoles per uL of reaction volume) for all three.
FIGURE: #copPCR cycling conditions.
Merits
Easy and reliable detection of #PointMutations without any sophistication.
It is very #specific with the competition between primers favoring the #mismatch specific primer a 100 times more.
Less #hazardous alternative to #RadiolabelledASO (Allele Specific Oligonucleotides) based approaches.
The #copPCR primers can also be used as #ASOprobes.
#copPCR can also be performed by #RealTimePCR providing a faster alternative with deferentially labeled primers.
Demerits
#Oligonucleotides has to be properly #purified with #SwiftLC or #HPLC purification recommended.
Purification of #oligos can increase the cost a bit.
This can only be used in cases of #KnownMutations.
Applications
Detection of various #GeneticDisorders caused by #PointMutations.
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