We're trying to measure relative telomere length (or at least T/S ratios) using the original technique of Cawthon (PMID:12000852). This seems to be about the only technique that people are currently reporting in the literature.
We have been through all the usual troubleshooting steps, and are getting nothing but primer dimers for the Tel1/Tel2 primer pair. The PCR for the single copy gene works wonderfully, and we know we have good quality DNA.
Does anybody have any experience with this particular technique? and are there any tips/tricks to getting it working?
Thanks
Jon
we collaborated with Elissa Epel and Liz Blackburn at UCSF. Why don´t you contact them. Liz is now president of Salk. Cheers, Dirk
Thanks Dirk.
I e-mailed Robby with the same question as he was also an author on your 2011 PNAS paper. If he can't answer then I will try Elissa Epel.
Thanks
Jon
Update: The problem has been solved.
We annealed to two primers together in the absence of enzyme or template, and ran them on a gel as a control for primer dimers. We actually had the correct product as our PCR band was above the primer dimers.
qPCR provides an easy and accurate way to measure telomere length. You simply needs 2 pairs of primers, one to amplify telomere and the other to amplify a single copy reference on the genome. However, you need to make sure 1) both primer pairs efficiency is close to 100%; 2) no non-specific amplification for both primer pairs, and 3) no primer dimer formation (especially for telomere primer pair) within 30 PCR cycles.
https://www.sciencellonline.com/catalogsearch/result/?q=telomere
https://www.sciencellonline.com/PS/8918.pdf
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