Methods for the detection of nucleic acid differences
AssigneeThe Johns Hopkins University
InventorsJames Eshleman, Chanjuan Shi, Susan Eshleman
Life of this patent
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Abstract
The invention provides methods permitting the detection of small amounts of different nucleic acids in the presence of an excess amount of wild-type nucleic acids. Also provided herein are method so detecting infectious disease minority variants, methods of forensic identification, methods of diagnosing cancer and monitoring disease progress.
Claims28
A method of detecting infectious disease minority variants, comprising:
- contacting a nucleic acid sample from a subject or a cell with at least one oligonucleotide pair to form a reaction mixture, wherein a pair comprises a first (P1) and a second (P2) oligonucleotide, wherein P1 comprises a first gene specific region and a first primer region, P2 comprises a second gene specific region and a second primer region;
- either P1 or P2 comprise a detection region;
- either P1 or P2 comprise a nucleotide difference in the gene specific region;
- and P1 and P2 are suitable for ligation to one another;
- subjecting the reaction mixture to a ligation reaction;
- and
- amplifying a ligation product to form a reaction product.
A method of differentiating pancreatic cancer from chronic pancreatitis comprising:
- contacting a nucleic acid sample from a subject or a cell with at least one oligonucleotide pair to form a reaction mixture, wherein a pair comprises a first (P1) and a second (P2) oligonucleotide, wherein P1 comprises a first gene specific region and a first primer region, P2 comprises a second gene specific region and a second primer region;
- either P1 or P2 comprise a detection region;
- either P1 or P2 comprise a nucleotide difference in the gene specific region;
- and P1 and P2 are suitable for ligation to one another;
- subjecting the reaction mixture to a ligation reaction;
- and
- amplifying a ligation product to form a reaction product.
A method of diagnosing a disease in a subject comprising:
- obtaining a biological sample from a subject;
- contacting the biological sample with at least one oligonucleotide pair to form a reaction mixture, wherein a pair comprises a first (P1) and a second (P2) oligonucleotide, wherein P1 comprises a first gene specific region and a first primer region, P2 comprises a second gene specific region and a second primer region;
- either P1 or P2 comprise a detection region;
- either P1 or P2 comprise a nucleotide difference in the gene specific region;
- and P1 and P2 are suitable for ligation to one another;
- subjecting the reaction mixture to a ligation reaction;
- and
- amplifying a ligation product to form a reaction product.
16-22. (canceled)
A method of forensic identification, comprising:
- obtaining a nucleic acid forensic sample;
- contacting a nucleic acid forensic sample with at least one oligonucleotide pair to form a reaction mixture, wherein a pair comprises a first (P1) and a second (P2) oligonucleotide, wherein P1 comprises a first gene specific region and a first primer region, P2 comprises a second gene specific region and a second primer region;
- either P1 or P2 comprise a detection region;
- either P1 or P2 comprise a nucleotide difference in the gene specific region;
- and P1 and P2 are suitable for ligation to one another;
- subjecting the reaction mixture to a ligation reaction;
- and
- amplifying a ligation product to form a reaction product.
24-27. (canceled)
A method of identifying a sample containing an uncommon genetic change, comprising:
- obtaining a pooled nucleic acid sample;
- contacting the pooled sample with at least one oligonucleotide pair to form a reaction mixture, wherein a pair comprises a first (P1) and a second (P2) oligonucleotide, wherein P1 comprises a first gene specific region and a first primer region, P2 comprises a second gene specific region and a second primer region;
- either P1 or P2 comprise a detection region;
- either P1 or P2 comprise a nucleotide difference in the gene specific region;
- and P1 and P2 are suitable for ligation to one another;
- subjecting the reaction mixture to a ligation reaction;
- and
- amplifying a ligation product to form a reaction product.
31-39. (canceled)
A method of detecting a nucleic acid sequence difference, comprising:
- contacting a nucleic acid sample with at least one oligonucleotide pair, wherein a pair comprises a first (P1) and a second (P2) oligonucleotide, wherein P1 and P2 bind to a nucleic acid to form a reaction mixture, and wherein P1 comprises a first gene specific region and a first primer binding region and P2 comprises a probe binding region, a second primer binding region and a second gene specific region, wherein there is a gap of one or more nucleotides between the first and second gene specific regions contains the nucleic acid difference;
- providing the reaction mixture with a nucleotide complementary to the nucleotide differencenucleic acid difference;
- subjecting P1, P2, and the nucleotide to ligation conditions to form a ligation product;
- and
- amplifying the ligation product to form a reaction product.
43-74. (canceled)
A kit for one of more of detecting infectious disease minority variants, differentiating pancreatic cancer from chromic pancreatitis, diagnosing a disease, forensic identification, comprising:
- at least one first (P1) and at least one second (P2) oligonucleotide to form a reaction mixture, wherein P1 comprises a first gene specific region and a first primer region, P2 comprises a second gene specific region and a second primer region;
- either P1 or P2 comprise a detection region;
- either P1 or P2 comprise a nucleotide difference in the gene specific region;
- and P1 and P2 are suitable for ligation to one another;
- and
- instructions for use.
A kit for one of more of detecting infectious disease minority variants, differentiating pancreatic cancer from chromic pancreatitis, diagnosing a disease, forensic identification, or detecting a nucleic acid sequence difference, comprising:
- at least one first (P1) and at least one second (P2) oligonucleotide, wherein P1 and P2 bind to a nucleic acid to form a reaction mixture, and wherein P1 comprises a first gene specific region and a first primer binding region and P2 comprises a probe binding region, a second primer binding region and a second gene specific region, wherein there a nucleotide gap between the first and second gene specific regions contains the nucleic acid difference;
- and
- instructions for use.
Dependencies are inferred from the claim text.