GAA repeat expansion of Friedreich's ataxia (FA) - How to identify and size expansion? (Jan/13/2007 )

Hello. I dig this forum, but I'm new to laboratory techniques used in molecular biology. My school is only a two-year college, but the bio lab is purchasing a thermocycler soon. I'm a biology student that follows research associated with the genetic disease Friedreich's ataxia (FA). Basically, I'd like to learn the procedures used in diagnosis of FA (via PCR), so I can verify results from a 5-year old test. If anyone doesn't mind, could you glance at my links? Some clarification would be very helpful.

After I extract DNA from a whole blood sample (source), I should follow standard PCR protocol (which was mentioned here in methods). Another one of my references is this. Is there a particular sort of kit I should annoy my prof about? And how do I obtain those primers? Hmm, I think that's it for now. Thanks!

You chose a rather difficult target for a first time PCR experiment. I'd start with something easier. The problems with this are repetitive sequence, long sequence, poor starting material, and competition from the normal, shorter allele. If you do want to do it, I'd recommend that you follow the long PCR protocols, even though the fragments are not particularly long, since you require robust amplification of longer fragments in the presence of shorter ones which will normally amplify much more readily. The primers are easy to order, and should cost less than $50 from places such as IDT (www.idtdna.com). You don't want to be doing this as a substitute for competent clinical analysis.

Thanks for the reply. Actually, I have results that tested the same individual (via PCR tech) about 5 years ago. I'm relying on those results to compare the accuracy of my first PCR experiment. Because those findings showed a GAA triplet repeat expansion for both alleles (~650 & ~900), should I still expect amp. competition from the shorter allele? Or would that usually be an issue for testing heterozygotes? Thanks again.

oh , and there is the problem with PCR slipage.
Since this is a repeat of the same 3 bp over and over again. It is very posible for a small segment of the PCR template to loop out, , a partially finished product to bridge the loop, and the polymerase continues to extend.. this result in a truncated PCR product. And because it is smaller it will be more competitive during the amplification.

It was quite a problem with a friend of mind who was working on triplet expension... the PCR product was a terrible smear. Reducing the extension temperature helped some. 68 Celsius.