I am working on Amyotrophic lateral sclerosis and try to amplify the GGGGCC hexanucleotide repeat expansion on Chromosome 9 (C9ORF72). I amplified it with FAM labelled primers by repeat primed PCR and ran the product on sequencer, but I did not get exactly six nucleotide differences between peaks. I also have done normal PCR with outer primers and got smaller sized products. Please help me out if anyone is working with repeat primed PCR Thanks.
Could there be a polymorphism in this particular sample? Do you have a different sample to demonstrate whether it is your technique that has a problem or the sequence of the sample you are testing.
Difficult to give any meaningful answer. What protocols have you used? Any of the published ones for C9ORF72?
These GC-repeats are notoriously difficult to amplify and polymerase slippage is basically normal. But to give any answer one would really need to know the primers etc. and the conditions that were used.
Thanks all
I am using the primers conditions from published article Ranton et al. 2011.
And yes i have used it with two different control samples.
dear all If I will be able to amplify this region with outside primers than is it feasible to sequence by sanger method.
Asuragen recenty launched a PCR based assay for C9orf72 amplification. You can find more information here: http://asuragen.com/portfolio/genetics/amplidex-pcrce-c9orf72/
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