Hello all, I am trying to do a PCR for a repeat rich region (AT rich) which gets expanded. I have tried using normal Thermo Taq Pol, LA taq from Takara. However I am not able to amplify the longer product. The wild type allele is getting amplified which does not undergo expansion. Can someone please help me to standardize the PCR conditions
what are the sizes of the WT and the expanded amplimer and what is the single repeat sequence please?
Hi Sir
The WT is 509 bp. Expanded amplimer size is approx 2kb and the repeat sequence is (TTTTA)7(TTA)(TTTTA)13.
That does not seem too long an amplimer . You need to minimise secondary structure effects and maximise amplification of the longer fragment so generally..
Use primers with high annealing temperatures ( over 60c) to minimise structural problems. Run a gradient of annealing temperatures to get the highest annealing temperature possible again to minimise re annealing. Keep the extension times long to maximise the long amplimer...try 3minutes extension. Keep the annealing time short...10seconds is plenty.
Run a sample expected to have the expansion at a temperature that works on the short amplimer but add increments of DMSO at 1,2,3,4,5,6% DMSO to minimise structural problems. If all of that fails then do the same but with a different taq polymerase
Hi, Rahul, first of all, have you confirmed that there is the target repeat expansion in your patients and by what methods? If you have confirmed, do you have a positive control with limited length of repeat expansion like 5-6kb? As reported, the expansion could be as long as 18kb. If your patients' repeat expansion was longer than 10kb, long-range PCR can not detect it.
Zhidong Cen Thank you so much for your reply, actually we are working to find the answer for the same so we want to check the range the repeat expansion by the means of long range PCR.
Rahul I agree with all the answer you received. One thing is sure the length that you have said is not too long to matter any way. However, if you still want to have more knowledge, I am giving a reference of an article which might help you.
SCIENTIFIC REPORTS | 4 : 5737 | DOI: 10.1038/srep05737
- Badges
- Science method