Compositions and methods for polynucleotide sequence detection
AssigneeStratagene
InventorsJoseph A. Sorge, Andrew Firmin
Life of this patent
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Abstract
The present invention provides compositions, kits, and methods for detecting polynucleotide sequence differences. The method involves amplifying a polynucleotide in the presence of a labeled nucleotide whose incorporation into the amplified product can indicate the presence of a sequence difference within the polynucleotide template. The invention is particularly useful for differentiating two or more closely related polynucleotide sequences, for example, in determining which allele or alleles of a multiallelic organism are present in a target polynucleotide.
Claims61
A method for determining a sequence difference between a region of interest in a polynucleotide and a reference sequence, said method comprising:
- a) incubating said polynucleotide in a reaction mixture comprising a nucleotide labeled with a detectable label to produce a polynucleotide product from said polynucleotide;
- b) determining an incorporation frequency of said labeled nucleotide for said polynucleotide product;
- and c) comparing said incorporation frequency determined in step (b) with a known frequency for said reference sequence, wherein a difference in the two frequencies is indicative of a sequence difference between the region of interest of said polynucleotide and said reference sequence.
The method of claim 4, wherein said polynucleotide product is linked to a solid support.
The method of claim 5, wherein the number of potential linkage sites on each specimen of said solid support is substantially constant and said linked polynucleotide product saturates the potential linkage sites on said solid support.
The method of claim 4, wherein said amount of the polynucleotide product is measured by polynucleotide staining with a polynucleotide stain.
A method for determining a sequence difference between a region of interest in a first polynucleotide and a corresponding region of interest in a second polynucleotide, said method comprising:
- a) incubating said first polynucleotide in a first reaction mixture comprising a nucleotide labeled with a detectable label to produce a first polynucleotide product from said first polynucleotide;
- b) determining an incorporation frequency of said labeled nucleotide for said first polynucleotide product;
- and c) comparing said incorporation frequency determined in step (b) with an incorporation frequency for said second polynucleotide, wherein a difference in the two incorporation frequencies is indicative of a sequence difference between the region of interest of said first polynucleotide and the corresponding region of interest of said second polynucleotide.
A method for determining the presence of a mutation in a region of interest in a polynucleotide, said method comprising:
- a) incubating said polynucleotide in a reaction mixture comprising a nucleotide labeled with a detectable label, to produce a polynucleotide product from said polynucleotide;
- b) determining an incorporation frequency of said labeled nucleotide for said polynucleotide product;
- and c) comparing said incorporation frequency determined in step (b) with a known frequency for a reference wild-type sequence, wherein a difference in the two frequencies is indicative of the presence of a mutation in a region of interest in a polynucleotide.
A method for genotyping comprising:
- a) incubating in a reaction mixture a first nucleic acid sample comprising a region of interest, said reaction mixture comprising a nucleotide labeled with a detectable label to produce a polynucleotide product from said nucleic acid sample;
- and b) determining an incorporation frequency of said labeled nucleotide for said polynucleotide product, wherein said ascertained incorporation frequency is indicative of the genotype of the organism from which said nucleic acid sample was obtained.
Dependencies are inferred from the claim text.