Wretched dinucleotide repeats

John Wolff wolffie at u.washington.edu
Mon Jun 10 12:59:33 EST 1996


In article <4okhac$ljk at sol.sun.csd.unb.ca>, sgriffit at rpc.unb.ca (Steve
Griffiths) wrote:

>  We're using selected bovine microsattelites as tools in forensic 
> analysis of moose and white tailed deer material to catch poachers.As 
> with any dinucleotide repeat system the real allele holds court to an 
> xtra band(s?) as a result of T addition by Taq ,as well as the lower 
> molecular weight stutter bands that I'm lead to believe result from the 
> collapse or shift of repeat units.I've been dickering around with T4 
> polymerase which is supposed to nip the T overhangs off the amplified 
> product, but ,so far,the enzyme and 5X buffer supplied by Gibco don't 
> appear to have much effect. Has anyone had any success in reducing the 
> appearance of these additions or shortened products? Is there any way of 
> preventing the stutter effect?

If you can find polymorphic tri- or tetra-nucleotide repeats, they're a
lot easier to read.  I've done quite a few different human dinucs and
noticed a lot of variability.  Some just look downright weird.  Sometimes
with tri- or tetra-nuc repeats, you have to look out for PCR apparently
amplifying the smallest allele preferentially -- the smallest band is
always darker, and sometimes the larger allele fails to show.

BUT:  the stutter bands are a nuisance, not an insuperable problem.  The
hardest part is to tell a homozygote from a heterozygote whose alleles are
just 2bp different:  in such a heterozygote, the 2nd largest band will be
DARKER than the largest, while in a homozygote, the largest band should be
darker.  You have to be REALLY careful.  I'm sure defense lawyers would
have a great time with these things!!



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