Methods for the Reduction of Stutter in Microsatellite Amplification

Abstract

The invention provides a method for reducing stutter in the amplification of a microsatellite comprising the steps of providing a sample comprising a microsatellite having a G+C content of 50% or less; contacting the sample with at least one enzyme having nucleic acid polymerase activity; and incubating the sample with the enzyme for a sufficient amount of time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of betaine, sorbitol or mixtures thereof, effective to reduce stutter relative to the amount of stutter observed in the absence of betaine and/or sorbitol. The invention also provides compositions containing betaine and/or sorbitol, kits for amplifying microsatellites having a G+C content of 50% or less, and methods of using all of the foregoing.

1 . A kit for the amplification of a target nucleic acid sequence, said target nucleic acid sequence comprising a microsatellite selected from the group consisting of mononucleotide microsatellites, dinucleotide microsatellites, tetra nucleotide microsatellites and pentanucleotide microsatellites, said microsatellite having a G+C content of 50% or less, comprising, in separate containers:
 a polymerase,   a plurality of deoxynucleotide triphosphates; and   an additive   
       wherein said additive is selected from the group consisting of sorbitol, betaine and mixtures thereof. 
     
     
         2 . The kit of  claim 1  wherein said polymerase is selected from the group consisting of AmpliTaq Gold® DNA polymerase; AmpliTaq® DNA Polymerase; AmpliTaq® DNA Polymerase, Stoffel fragment; rTth DNA Polymerase; rTth DNA Polymerase XL; Tne, Bst DNA polymerase large fragment from  Bacillus stearothermophilus ; Vent and Vent Exo- from  Thermococcus litoralis; Tma  from  Thermotoga maritiama ; Deep Vent and Deep Vent Exo- and Pfu from  Pyrococcus ; and mutants, variants and derivatives thereof.