"Stutter" peaks are an inherent part of PCRing repeat based markers.
Shannon Odelberg here at the U of U showed that you CAN get rid of the
stutter peaks by amplifying linearly with one primer for 50-100 cycles, then
purifying your product to get rid of that primer and amplifying with the
other primer for 50-100 cycles. But who's going to do that??!! He had a
couple of papers explaining how stutter bands are produced--you should be
able to find them through Medline if you are interested.
Mono-nucleotides are worst to the point of impossible, di-nucs are pretty
bad, tri- and tetras are pretty good. Additionally, the more repeats there
are, the worse the ladder is, which is sad because more repeats make for a
more polymorphic marker. Most mono-nucs and a few di-nucs have higher as
well as lower stutter bands, but the di-nuc higher bands are generally small
enough not to be confusing.
We have used the PE MD10 kit which is all di-nucs and have found it to be
pretty good. Of course, I remember having to read auto-rads and believe me
it is easier to look at peak heights than trying to decide if one black spot
is darker than another! We did find that about 5% of the markers had some
alleles 1bp different from another and that is always a worry, particularly
if you have multiple gels.
Good luck,
Linda
> ----------
> From: sharon.palmer@spectrum-health.org
> Sent: Wednesday, February 9, 2000 9:50 AM
> To: Recipients of ABRF List
> Subject: linkage mapping
> Importance: High
>
> Has anyone "played" with the PE linkage mapping kit(s)? Does anyone have
> a
> protocol to minimize the amount of stutter?
>
> Any assistance would be greatly appreciated.
>
> Sharon Keely Palmer
> Spectrum Health
> Phone 616.356.4062
> fax 616.356.4035
>
This archive was generated by hypermail 2b29 : Mon Feb 14 2000 - 16:05:59 EST