Stutter Bands

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Tue Jul 23 12:04:27 EST 1996


David Groth (igrothd at INFO.CURTIN.EDU.AU) wrote:

> Can anyone suggest a cure for stutter bands when analysing 
> dinucleotide microsatellites by PCR.  We are developing several 
> microsatellites for use in population studies in a fresh water 
> crustacean.  The loci identified so far all have very large repeat 
> regions eg CA(50-70 repeats). These are usually perfect although they 
> can be complex.  

> When we do PCR we get 5-8 bands displaced 2bp in a ladder formation.  
> This is a greater problem than I have seen in other species eg human.  
> We have tried using DMSO, increasing the annealing temp, Taq gold 
> without removing the problem. I have thought of using deaza-GTP 
> instead of dGTP.  Has anyone tried this?

You might want to look into using a different buffer for the PCR:

@article{Woodford1995,
author = "K. Woodford
     and M. N. Weitzmann
     and K. Usdin",
title = "The use of {K+}--free buffers eliminates a common
cause of premature chain termination in {PCR} and {PCR} sequencing",
journal = "Nucleic Acids Res.",
volume = "23",
number = "3",
pages = "539",
year = "1995"}

@article{Woodford1994,
author = "K. J. Woodford
     and R. M. Howell
     and K. Usdin",
title = "A novel {K+}--dependent {DNA} synthesis arrest site
in a commonly occurring sequence motif in eukaryotes",
journal = "J. Biol. Chem.",
volume = "269",
number = "43",
pages = "27029-27035",
month = "oct",
year = "1994"}
 
@article{Usdin1995,
author = "K. Usdin
     and K. J. Woodford",
title = "{CGG} repeats associated with {DNA} instability
and chromosomal fragility form structures that block {DNA}
synthesis {\em in vivo}",
journal = "Nucleic Acids Res.",
volume = "23",
number = "20",
pages = "4202-4209",
year = "1995"}

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