Don't go literal, it doesn't get delayed! The Linear After The Exponential PCR (#LATEPCR) is an efficient way of surpassing the #PCRplateau and achieving greater sensitivity and specificity for #qPCR based assays as well as a steady production of #ssdNA to be further used on various applications.
Principle
In conventional PCR, the reaction slows down after the initial exponential phase and plateaus #stochastically because of #reannealing of the amplicon strands, leaving lesser chances for primer or probe to bind and carry on with the exponential phase. This problem can be solved with #nonequimolar concentrations of the primers as is done in #asymmetricPCR, but that too is disadvantageous with various optimizations.
LATE PCR comes to rescue here with modified primer designing and certain cycling tweaks. As with asymmetric PCR, use of unequal amounts primers gives an edge to the excess primer over limiting primer in amplifying the strand of choice after the initial exponential phase. Use of low Tm #molecularprobes in qPCRs, increases specificity and sensitivity of the reaction by uncoupling the probe-binding and primer-annealing step i.e., including a short step for probe binding after the extension step at a lower temperature.
FIGURE: A schematic representation of the #LATEPCR thermal cycling for both #ConventionalPCR and #qPCR based approaches.
Protocol
The protocol changes from conventional PCR/ qPCR in following contexts:
a. Primer designing: It has two rules, (TmL–TmX) ≥5°C and (TmA–TmX) ≤18°C, where TmL, TmX and TmA are the melting temperatures of #LimitingPrimer, #eXcessPrimer, and #AmpliconStrands.
b. Primer ratio: The recommended ratio of #LimitingPrimer to #eXcessPrimer is 1:20, although variations of 10-50 folds can be used.
c. Low Tm probes: Use of low Tm molecular beacons in conjunction with other modifications greatly enhances the signal strength of the assays. The molecular beacon loop Tm has to be 5-10°C below the Tm of the limiting primer (TmL).
d. PCR enhancers: Certain reactions demand enhancers and 1 mg/ml BSA or 750 mM trehalose or 1% Tween-20 can be used in LATE PCR reactions.
Merits
It maintains the #PCRefficiency of #symmetricPCR while bringing on the benefits of #asymmetricPCR.
It increases the signal strength of Probe based qPCR by 80-250% relative to even the #symmetricPCR.
It increases probe reliability in #qPCR by uncoupling the primer annealing and probe binding step, thereby creating chances for the use of low Tm probes and thus better signal strength and signal-to-noise ratio.
Low Tm probes being smaller in size, has less tolerance towards mismatch and thus greater specificity for detection of #amplicons. They can be used in high concentrations as well as there is no competition with primers of higher Tm.
It shows efficient #LinearKinetics even for >80 cycles.
Ideal for #LabOnAChip format.
Compatible with existing thermal cyclers.
Demerits
It takes too long (~80 cycles) to yield considerable #ssDNA.
Applications
One of the most used applications of #LATEPCR is determining the #zygosity of several genetic disorders through low Tm #molecularbeacon based #qPCR.
The efficient production of #ssDNA can be channelled to #pyrosequencing, probes for #microarrays, single-strand conformation polymorphism analysis (#SSCP), single-nucleotide polymorphism (#SNP) screening, and #digitalPCR through automated #RaceTrackReactionChamber.
#DNAaptamer production is another application.
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