I have gone through multiple research article for TP PCR based diagnosis of Friedreich ataxia. the majority of paper who shows the basic concept of tp pcr are telling to label target specific forward or reverse primer. and beside that target specific primer, there will be more two primers, one will be universal sequence and another will be universal + repeat sequence. here in these reaction, Universal + repeat sequence primer will be in very lesser concentration where other 2 will be on same concentration. so based on TP PCR principle, it can produce specific pattern for expanded alleles.

Here i got the primer sequence from well known and well cited article (below) and is stating that they label the Universal + repeat sequence primer.

"Schmitt M, Warner J, Barron L, Mandel J, M Koenig. Detection of large (GAA)n repeat expansions by fluorescent PCR, 2000, European Society for Human Genetics Poster presentation."

"Ciotti P, Di Maria E, Bellone E, Ajmar F, Mandich P. Triplet repeat primed PCR (TP PCR) in molecular diagnostic testing for Friedreich ataxia. J Mol Diagn. 2004 Nov;6(4):285-9. doi: 10.1016/S1525-1578(10)60523-5. PMID: 15507666; PMCID: PMC1867489.

Primer seq:

Triplet repeat PCR (TP-PCR)

Schmitt et al (contact M Koenig for further details)

· P1 5'-GCTGGGATTACAGGCGCGCGA-3' - target based reverse seq

· P3 5'-TACGCATCCCAGTTTGAGACG-3' - universal seq

· P4 5'-TACGCATCCCAGTTTGAGACGGAAGAAGAAGAAGAAGAAGAA- 3' - Univarsal + repeat seq primer with tagging FAM.

So i doubt the outcome for this set up.

Is there anybody is experienced of using same primers with same tagging to same primer then please guide.

If is there any better way or better update on this FRDA diagnosis then please guide.

If is anybody who can guide me better on this then please guide for the same.

Thank you for your time and efforts.