Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-07
2004-08-24
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S092000, C536S023100, C536S024300
Reexamination Certificate
active
06780588
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods, compositions and kits for reducing stutter in polymerase chain reaction amplification of microsatellites. In certain embodiments, the invention relates to the use of sorbitol in polymerase chain reactions in an amount effective to reduce stutter in the amplification of mononucleotide, dinucleotide, trinucleotide, tetranucleotide and pentanucleotide microsatellites.
BACKGROUND OF THE RELATED ART
Microsatellites, or short tandem repeats (STRs), consist of tandemly repeated DNA sequence motifs of 1 to 6 nucleotides in length. They are widely dispersed and abundant in the eukaryotic genome, and are often highly polymorphic due to variation in the number of repeat units. This polymorphism renders microsatellites attractive DNA markers for genetic mapping, medical diagnostics and forensic investigation. The combination of PCR and gel or capillary electrophoresis under denaturing conditions has greatly improved the genotyping of microsatellite DNA sequences. However, PCR artifacts exhibited by non-proofreading enzymes and referred to as stutter and the terminal transferase side-reaction can complicate analysis of closely spaced microsatellite alleles.
Stutter signals differ from the PCR product representing the genomic allele by multiples of repeat unit size. For dinucleotide repeat loci, the prevalent stutter signal is generally two bases shorter than the genomic allele signal, with additional side-products that are 4 and 6 bases shorter. The multiple signal pattern observed for each allele especially complicates interpretation when two alleles from an individual are close in size (e.g., medical and genetic mapping applications) or when DNA samples contain mixtures from two or more individuals (e.g., forensic applications). Such confusion is maximal for mononucleotide microsatellite genotyping, when both genomic and stutter fragments experience one-nucleotide spacing.
There is a need in the art to develop PCR reaction conditions that minimize or eliminate stutter so that genetic analysis may be more accurate and reliable. This invention is directed to these, as well as other, important ends.
SUMMARY
In accordance with some embodiments of the methods of the invention, methods for reducing stutter in the amplification of a microsatellite are provided comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite;
wherein said incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
The invention also provides methods for reducing stutter in the amplification of a mononucleotide microsatellite comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite;
wherein the incubation is performed in the presence of an amount of sorbitol, wherein the sorbitol is effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
The invention also provides methods for reducing stutter in the amplification of a dinucleotide microsatellite comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
The invention further provides methods for reducing stutter in the amplification of a trinucleotide microsatellite comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
The invention further provides methods for reducing stutter in the amplification of a tetranucleotide microsatellite comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
The invention further provides methods for reducing stutter in the amplification of a pentanucleotide microsatellite comprising the steps of:
(a) providing a sample comprising a microsatellite of interest, in which the microsatellite has a G+C content of greater than 50%;
(b) contacting the sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating the sample with the enzyme for a time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol.
In further embodiments of the methods of the invention, methods are provided comprising the steps of:
(a) providing a sample comprising a nucleic acid that contains one or more microsatellites selected from the group consisting of mononucleotide microsatellites, dinucleotide microsatellites, trinucleotide microsatellites, tetranucleotide microsatellites and pentanucleotide microsatellites; and
(b) amplifying at least one nucleobase sequence of said nucleic acid, said nucleobase sequence comprising at least one of said microsatellites; said amplified microsatellite having a G+C content of greater than 50%; wherein said amplification is performed in the presence of sorbitol.
Also provided in accordance with the present invention are methods for performing polymerase chain reaction amplification of a microsatellite selected from the group consisting of mononucleotide microsatellites, dinucleotide microsatellites, trinucleotide microsatellites, tetranucleotide microsatellites and pentanucleotide microsatellites, said microsatellite having a G+C content of greater than 50%; said method comprising the step of contacting said microsatellite with a polymerase in the presence of an amount of sorbitol effective to reduce the amount of stutter arising from said amplification relative to the amount of such stutter observed in the absence of sorbitol.
Also provided by the present invention are methods of detecting cancer or a pre-cancerous condition and genetic disorders, 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
Bloch Will
Coticone Sulekha Rao
Bortner Scott R.
Finn Andrew K.
Fredman Jeffrey
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