Nucleic acid detection combining amplification with fragmentation
First Claim
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1. A method for diagnosing an individual with cancer by determining if an individual has a mutant nucleic acid associated with the cancer, said method comprising:
- a) providing a sample comprising unamplified nucleic acids from the individual, wherein said unamplified nucleic acids potentially contain the mutant nucleic acid in the presence of non-mutant nucleic acid, wherein said mutant nucleic acid lacks at least one fragmentation site present in a non-mutant nucleic acid and said mutant nucleic acid and said non-mutant nucleic acid are at least 50% identical at aligned nucleotide positions;
b) fragmenting the unamplified nucleic acids under conditions such that a subsequent amplification directed to the mutant nucleic acid results in an increased detection of said mutant nucleic acid over said non-mutant nucleic acid as compared to amplification without fragmentation;
c) combining the unamplified nucleic acids of step b) with a polymerase chain reaction (PCR) mixture;
d) amplifying the mutant nucleic acid from the unamplified nucleic acids combined with the PCR mixture in step c) with a pair of primers specific for the mutant nucleic acid, wherein one primer spans or overlaps the at least one fragmentation site present in the non-mutant nucleotide sequence but absent from the mutant nucleotide sequence, and wherein fragmentation of the non-mutant nucleotide sequence in part b) prevents hybridization of said one primer to the non-mutant nucleic acid; and
e) detecting the presence or absence of an amplification product from step d) containing the mutant nucleic acid, wherein diagnosis of cancer is determined by the presence absence or amount of amplification product containing the mutant sequence;
wherein the non-mutant nucleic acid comprises a wild-type nucleotide sequence and the mutant nucleic acid comprises a mutated version of the wild-type nucleic acid sequence, and wherein step b) is performed prior to step c).
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Abstract
Provided herein are methods and compositions for detection of a nucleic acid target in a sample. The methods and compositions use primer directed amplification in conjunction with nucleic acid fragmentation. The methods have high sensitivity even in the presence of a large amount of non-target nucleic acid. Also provided are oligonucleotides and kits useful in the method. Exemplary nucleic acid targets are those with mutant gene sequence such as mutant sequence of the EGFR, APC, TMPRSS2, ERG and ETV1 genes.
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Citations
20 Claims
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1. A method for diagnosing an individual with cancer by determining if an individual has a mutant nucleic acid associated with the cancer, said method comprising:
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a) providing a sample comprising unamplified nucleic acids from the individual, wherein said unamplified nucleic acids potentially contain the mutant nucleic acid in the presence of non-mutant nucleic acid, wherein said mutant nucleic acid lacks at least one fragmentation site present in a non-mutant nucleic acid and said mutant nucleic acid and said non-mutant nucleic acid are at least 50% identical at aligned nucleotide positions; b) fragmenting the unamplified nucleic acids under conditions such that a subsequent amplification directed to the mutant nucleic acid results in an increased detection of said mutant nucleic acid over said non-mutant nucleic acid as compared to amplification without fragmentation; c) combining the unamplified nucleic acids of step b) with a polymerase chain reaction (PCR) mixture; d) amplifying the mutant nucleic acid from the unamplified nucleic acids combined with the PCR mixture in step c) with a pair of primers specific for the mutant nucleic acid, wherein one primer spans or overlaps the at least one fragmentation site present in the non-mutant nucleotide sequence but absent from the mutant nucleotide sequence, and wherein fragmentation of the non-mutant nucleotide sequence in part b) prevents hybridization of said one primer to the non-mutant nucleic acid; and e) detecting the presence or absence of an amplification product from step d) containing the mutant nucleic acid, wherein diagnosis of cancer is determined by the presence absence or amount of amplification product containing the mutant sequence; wherein the non-mutant nucleic acid comprises a wild-type nucleotide sequence and the mutant nucleic acid comprises a mutated version of the wild-type nucleic acid sequence, and wherein step b) is performed prior to step c). - View Dependent Claims (2, 6, 7, 8, 9, 10)
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3. A method for prognosis of an individual diagnosed with cancer by determining if an individual has a mutant nucleic acid associated with the cancer, said method comprising:
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a) providing a sample comprising unamplified nucleic acids from the individual wherein said unamplified nucleic acids potentially contain the mutant nucleic acid in the presence of non-mutant nucleic acid, wherein said mutant nucleic acid lacks at least one fragmentation site present in a non-mutant nucleic acid and said mutant nucleic acid and said non-mutant nucleic acid are at least 50% identical at aligned nucleotide positions; b) fragmenting the unamplified nucleic acids under conditions such that a subsequent amplification directed to the mutant nucleic acid results in an increased detection of said mutant nucleic acid over said non-mutant nucleic acid as compared to amplification without fragmentation; c) combining the unamplified nucleic acids of step b) with a polymerase chain reaction (PCR) mixture; d) amplifying the mutant nucleic acid from the unamplified nucleic acids combined with the PCR mixture in step c) with a pair of primers specific for the mutant nucleic acid, wherein one primer spans or overlaps the at least one fragmentation site present in the non-mutant nucleotide sequence but absent from the mutant nucleotide sequence, and wherein fragmentation of the non-mutant nucleotide sequence in part b) prevents hybridization of said one primer to the non-mutant nucleic acid; and e) detecting the presence, absence and/or amount of an amplification product from step d) containing the mutant nucleic acid, wherein the likelihood of an outcome in said individual is associated with the presence and or amount of mutant nucleic acid sequence; wherein the non-mutant nucleic acid comprises a wild-type nucleotide sequence and the mutant nucleic acid comprises a mutant version of the wild-type nucleic acid sequence, and wherein step b) is performed prior to step c). - View Dependent Claims (4, 11, 12, 13, 14, 15)
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5. A method for determining drug sensitivity of an individual diagnosed with cancer, said method comprising:
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a) providing a sample comprising unamplified nucleic acids from the individual wherein said unamplified nucleic acids potentially contain the mutant nucleic acid in the presence of non-mutant nucleic acid, wherein said mutant nucleic acid lacks at least one fragmentation site present in a non-mutant nucleic acid and said mutant nucleic acid and said non-mutant nucleic acid are at least 50% identical at aligned nucleotide positions; b) fragmenting the unamplified nucleic acids under conditions such that a subsequent amplification directed to the mutant nucleic acid results in an increased detection of said mutant nucleic acid over said non-mutant nucleic acid as compared to amplification without fragmentation; c) combining the unamplified nucleic acids of step b) with a polymerase chain reaction (PCR) mixture; d) amplifying the mutant nucleic acid from the unamplified nucleic acids combined with the PCR mixture in step c) with a pair of primers specific for the mutant nucleic acid, wherein one primer spans or overlaps the at least one fragmentation site present in the non-mutant nucleotide sequence but absent from the mutant nucleotide sequence, and wherein fragmentation of the non-mutant nucleotide sequence in part b) prevents hybridization of said one primer to the non-mutant nucleic acid; and e) detecting the presence, absence and/or amount of an amplification product from step d) containing the mutant nucleic acid; and f) relating the presence, absence and/or amount of an amplification product containing the mutant nucleic acid to cancer drug sensitivity; wherein the non-mutant nucleic acid comprises a wild-type nucleotide sequence and the mutant nucleic acid comprises a mutant version of the wild-type nucleic acid sequence, and wherein step b) is performed prior to step c. - View Dependent Claims (16, 17, 18, 19, 20)
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Specification