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Old 01-27-2014, 08:32 AM   #1
JoeChris38
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Default PCR amplifying microsatellites (short tandem repeats) for sequencing

Hi all,

I'm looking to amplify specific dinucleotide repeats from low input DNA (~1ng) but I'm having problems with microsatellite contraction/expansion during rounds of PCR. Has anyone with experience in this area have a protocol that can amplify dinucleotide repeat tracts without contracting/expanding the repeat?

Thanks,

Joe
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Old 02-05-2014, 07:47 AM   #2
ATϟGC
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Hey Joe,

Minimizing microsat stutter is indeed one of the "holy grails" of PCR-based genotyping. I have tried numerous modifications to PCR myself to limit stutter in mononucleotide repeats with a bit of success. Among things that did not seem to work were: trehalose addition([~.2-1M]), PEG (~8%), betaine, formamide, minimized extension temperatures (to limit strand melting), minimized cycle number (<25). The only thing that I have found to ameliorate the PCR-induced stuttering in long A+T homopolymer tracks used in Sanger sequencing is sso7D-fused proofreading polymerases (http://www.biotechniques.com/Biotech...es-197505.html). I have also tried using sso7D-based polymerases on GC dinucleotide repeats (with 8 or more repeats) but I did not see any less stuttering compared to taq (compared on an acrylamide gel). I think that if you have less than 7 dinucleotide repeats that a sso7D-based proofreading polymerase might help as the repeat section would be less than 14 base pairs. Such polymerases might still be worth a shot with your loci as I didn't exhaustively test them as mononucleotide repeats were the main problem with my loci.

there is also a discussion over here (http://seqanswers.com/forums/showthr...hlight=chimera) about chimera formation that you might find interesting as some of the techniques used to minimize chimeras could help with stutter.

I hope this helps.
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Old 02-05-2014, 08:04 AM   #3
JoeChris38
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Thanks, that's really useful.

I noted this paper: http://www.ncbi.nlm.nih.gov/pubmed/?...+amplification showing that reduced temperature appeared to reduce stutter. Have you tried any isothermal amplification reactions at 37c?

I recently tried the NEB IsoAmp kit (https://www.neb.com/products/h0110-i...ersal-thda-kit) that works at 65c but this also caused stuttering...

Joe
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amplification, dinucleotides, low input, microsatellite, pcr

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