PCR amplification of large piece from Bissulfite treated DNA - (Nov/30/2006 )
Hello Everyone,
This is a very naive question, given that I am just starting to do this, but I was wondering why is it so hard to amplify large pieces of DNA (e.g. 3kb-8kb) from bisulfite treated DNA. Is it because with the treatment there is a lot of degredation or is there any other reasons. I appreciate your input. Thank you.
Regards,
Nael
Degradation is a major issue, but another problem is often the low GC content of converted DNA. If you have runs of 20+ bases nearly all AT, then you will have problems with PCR through those regions when the extension temperature is 72C. See
Su XZ, Wu Y, Sifri CD, Wellems TE.
Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acids Res. 1996 Apr 15;24(8):1574-5. PMID: 8628694
Thank you phage434
Su XZ, Wu Y, Sifri CD, Wellems TE.
Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acids Res. 1996 Apr 15;24(8):1574-5. PMID: 8628694