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 - 76 . (canceled) 
     
     
         77 . 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.   
     
     
         78 . The kit of  claim 77  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. 
     
     
         79 . A method of detecting cancer, a pre-cancerous condition or genetic disorder in a subject comprising amplifying a region of DNA from a subject, wherein said region comprises a microsatellite selected from the group consisting of a mononucleotide repeat, a dinucleotide repeat, a trinucleotide repeat, a tetranucleotide repeat, and a pentanucleotide repeat, wherein said amplification comprises the steps of:
 (a) providing a sample comprising a nucleic acid that contains a nucleic acid having a microsatellite instability,   (b) amplifying at least one nucleobase sequence of said nucleic acid, said nucleobase sequence comprising at least one of said microsatellites; and   (c) detecting alterations of said microsatellite as compared to corresponding microsatellites amplified from control tissue; said amplified microsatellite having a G+C content of 50% or less;   wherein said amplification is performed in the presence of a sufficient amount of an additive selected from the group consisting of sorbitol, betaine and mixtures thereof, wherein said additive is effective to reduce said stutter relative to the amount of stutter observed in the absence of said additive.   
     
     
         80 . The method of  claim 79  wherein said cancer or cancerous condition is selected from the group consisting of colorectal cancer and breast cancer. 
     
     
         81 . The method of  claim 79  wherein said genetic disorder is Friedreich's ataxia. 
     
     
         82 . The method of  claim 80  wherein said region comprises a genetic locus comprising A/T. 
     
     
         83 . The method of  claim 81  wherein said region comprises a genetic locus comprising GAA/TTC. 
     
     
         84 . The method of  claim 79  wherein said incubation is performed in the presence of a set of dNTPs, said set comprising an amount of dNTP complementary to adenosine, an amount of dNTP complementary to guanosine, an amount of dNTP complementary to cytidine and an amount of dNTP complementary to thymine; wherein each of said amounts of dNTP is least 0.5 mM. 
     
     
         85 . The method of  claim 79  wherein said incubation is performed in the presence of a set of dNTPs, said set comprising an amount of dNTP complementary to adenosine, an amount of dNTP complementary to guanosine, an amount of dNTP complementary to cytidine and an amount of dNTP complementary to thymine; wherein each of said amounts of dNTP is least 1 mM. 
     
     
         86 . The method of  claim 79  wherein said additive is present in an amount of 1.5 to 3.5 M. 
     
     
         87 . The method of  claim 79  wherein said additive is present in an amount of 2.0 to 3.0 M. 
     
     
         88 . A method of gene typing comprising amplifying a plurality of regions of DNA from a sample containing DNA from a subject, wherein said regions comprise at least one microsatellite selected from the group consisting of a mononucleotide repeat, a dinucleotide repeat, a trinucleotide repeat, a tetranucleotide repeat, and a pentanucleotide repeat, said microsatellite having a G+C content of 50% or less, wherein said amplification comprises the steps of:
 (a) contacting said DNA with a enzyme at least one enzyme having nucleic acid polymerase activity;   (b) incubating said sample with said enzyme for a time and under conditions sufficient to amplify said regions; and   (c) separating amplified regions, forming a microsatellite pattern;   wherein said incubation is performed in the presence of an amount of an additive selected from the group consisting of sorbitol, betaine and mixtures thereof, wherein said additive is effective to reduce said stutter relative to the amount of stutter observed in the absence of said additive.   
     
     
         89 . The method of  claim 88  wherein said incubation is performed in the presence of a set of dNTPs, said set comprising an amount of dNTP complementary to adenosine, an amount of dNTP complementary to guanosine, an amount of dNTP complementary to cytidine and an amount of dNTP complementary to thymine; wherein each of said amounts of dNTP is least 0.5 mM. 
     
     
         90 . The method of  claim 88  wherein said incubation is performed in the presence of a set of dNTPs, said set comprising an amount of dNTP complementary to adenosine, an amount of dNTP complementary to guanosine, an amount of dNTP complementary to cytidine and an amount of dNTP complementary to thymine; wherein each of said amounts of dNTP is least 1 mM. 
     
     
         91 . The method of  claim 88  wherein said additive is present in an amount of 1.5 to 3.5 M. 
     
     
         92 . The method of  claim 88  wherein said additive is present in an amount of 2.0 to 3.0 M. 
     
     
         93 . A method of personal genetic identification comprising amplifying a plurality of regions of DNA from a sample containing DNA from a subject, wherein said regions comprise at least one microsatellite selected from the group consisting of a mononucleotide repeat, a dinucleotide repeat, a trinucleotide repeat, a tetranucleotide repeat and a pentanucleotide repeat, wherein said microsatellite has a G+C content of 50% or less, wherein said amplification comprises the steps of:
 (a) contacting said DNA with a enzyme at least one enzyme having nucleic acid polymerase activity; and   (b) incubating said sample with said enzyme for a time and under conditions sufficient to amplify said regions;   (c) separating amplified regions, forming a microsatellite pattern; and   (d) comparing said microsatellite pattern with a corresponding microsatellite pattern derived from the a DNA sample from a second source;   wherein said incubation is performed in the presence of an amount of an additive selected from the group consisting of sorbitol, betaine and mixtures thereof, wherein said additive is effective to reduce said stutter relative to the amount of stutter observed in the absence of said additive.   
     
     
         94 . The method of  claim 93  wherein said incubation is performed in the presence of a set of dNTPs, said set comprising an amount of dNTP complementary to adenosine, an amount of dNTP complementary to guanosine, an amount of dNTP complementary to cytidine and an amount of dNTP complementary to thymine; wherein each of said amounts of dNTP is least 1 mM. 
     
     
         95 . The method of  claim 93  wherein said incubation is performed in the presence of a set of dNTPs, said set comprising an amount of dNTP complementary to adenosine, an amount of dNTP complementary to guanosine, an amount of dNTP complementary to cytidine and an amount of dNTP complementary to thymine; wherein each of said amounts of dNTP is least 1 mM. 
     
     
         96 . The method of  claim 93  wherein said additive is present in an amount of 1.5 to 3.5 M. 
     
     
         97 . The method of  claim 93  wherein said additive is present in an amount of 2.0 to 3.0 M.