John Ladasky wrote:
>> Greetings,
>> I'm doing a little microsatellite typing on the side of my regular
> project. I'm dismayed to see several prominent secondary products. My
> understanding is that these secondary peaks, called "stutter bands," arise
> from slipped-strand mispairing during PCR. A colleague said that using *two*
> temperature thermal cycling (anneal and extend at the same temperature) is
> supposed to help reduce the stutter bands, but he didn't remember the refer-
> ence. Can anyone vouch for this and/or remember the reference? Do you have
> any other hot tips for cleaning up microsatellite PCR? Thanks!
Two step PCR will minimize nontemplate nucleotide addition by Taq
(addition of extra A) - NOT stutter bands. Select tri- or
tetranucleotide repeats and you will see almost no stutter bands. The
ref is: Smith, Genome Res. 5:312-17 (1995).
Regards,
Flemming
--
Flemming Larsen
Dept. of Clin. Immunol., sect. 7631
National University Hospital
Tagensvej 20
DK-2200 Copenhagen N
Denmark
E-mail: flar at biobase.dk
phone: +45 35 45 78 48
fax: +45 31 39 87 66
