The #TriplePrimedPCR or #TripletRepeatPrimedPCR is a modified version of the #conventionalPCR aimed at specific and accurate enumeration of pathological repeat expansions in the DNA of #Huntingtons or #FriedrichAtaxia or #MyotonicDystrophy etc. patients.
Principle
As can be gauged from the name, this PCR uses 3 different primers: P1 that is a #fluorescent-labelled forward primer that sits just upstream of the repeat region, P4 that has 2-5 tandem repeats at its 3' end and a non-binding non-human unique sequence just after that till its 5'end, the P3 has this unique non-human tail sequence of the P4.
FIGURE: TP PCR schematics
In the initial amplification cycles, along with P1, the repeat specific reverse primer P4 anneals at multiple sites all along the #TripletRepeatRegion, amplifying #DNA fragments differing by one repeat producing a #3bpLadder.
In later amplification cycles, P1 and P3 amplify the PCR products produced by P1 and P4, preventing the gradual shortening of the average PCR product due to P4 priming at sites internal to the PCR products of earlier rounds. This maintains the specificity in amplification for the large pathogenic repeat sequences.
Protocol
The protocol is same as the conventional PCRs except the following:
1. Use of 3 different primers.
2. P1 and P3 primer concentration in the cocktail is usually kept 10 times more than P4.
3. A longer final extension time is provided, usually ~10 mins.
4. Some cases demand the use of #PCRenhancers!
FIGURE: General #TPPCR cycling conditions.
Merits
TP PCR has the ability to discriminate fully expanded pathological #alleles.
No #allelicdropout, as #TPPCR does not preferentially amplify smaller alleles like #conventionalPCR.
High #sensitivity and #specificity.
It takes lesser time compared to the classical analysis with #conventionalPCR followed by fragment length analysis and #SouthernBlot analysis.
Cheaper than the #PCR and #SouthernBlot based approach.
Demerits
It is often difficult to standardize the assay given the involvement of 3 different primers and aberrant annealing in the first few cycles over the repeat region.
It has also been seen that #LongRangePCR with #TthPolymerase and #Tricine Buffer works better in this protocol, marking it a not so cheap alternative to #SouthernBlot based approach.
Applications
Molecular diagnostics of nucleotide repeat expansion disorders like #FragileXSyndrome, #HuntingtonsDisease, #FriedreichAtaxia, #MuscularDystrophy etc.
Studies on #allelic heterozygosity.
More to analyse:
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