NUCLEIC ACID DETECTION COMBINING AMPLIFICATION WITH FRAGMENTATION
First Claim
1. A method for detecting the presence or absence of a target nucleic acid by testing a sample that potentially contains the target nucleic acid in the presence of non-target nucleic acid, said method comprising:
- a) fragmenting the sample nucleic acid under conditions such that a subsequent amplification directed to the target nucleic acid results in an increased detection of said target nucleic acid over said non-target nucleic acid as compared to amplification without fragmentation;
b) amplifying the target nucleic acid with a pair of primers, wherein a first primer is specific for the target nucleic acid; and
c) detecting the presence or absence of an amplification product, which indicates the presence or absence of the target nucleic acid in the sample.
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Accused Products
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
45 Claims
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1. A method for detecting the presence or absence of a target nucleic acid by testing a sample that potentially contains the target nucleic acid in the presence of non-target nucleic acid, said method comprising:
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a) fragmenting the sample nucleic acid under conditions such that a subsequent amplification directed to the target nucleic acid results in an increased detection of said target nucleic acid over said non-target nucleic acid as compared to amplification without fragmentation; b) amplifying the target nucleic acid with a pair of primers, wherein a first primer is specific for the target nucleic acid; and c) detecting the presence or absence of an amplification product, which indicates the presence or absence of the target nucleic acid in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)
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41. A method for diagnosing an individual with cancer by determining if an individual has a mutant sequence associated with the cancer, said method comprising:
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a) obtaining a sample comprising nucleic acid from the individual; b) fragmenting the sample nucleic acid 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) amplifying the mutant nucleic acid with a pair of primers, wherein a first primer is specific for the mutant nucleic acid; and d) detecting the presence or absence of an amplification product containing the mutant sequence, wherein diagnosis of cancer is determined by the presence absence or amount of amplification product containing the mutant sequence. - View Dependent Claims (42)
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43. A method for prognosis of an individual diagnosed with cancer by determining if an individual has a mutant sequence associated with the cancer, said method comprising:
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a) obtaining a sample comprising nucleic acid from the individual; b) fragmenting the sample nucleic acid 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) amplifying the mutant nucleic acid with a pair of primers, wherein a first primer is specific for the mutant nucleic acid; and d) detecting the presence, absence and/or amount of an amplification product containing the mutant sequence, wherein the likelihood of an outcome in said individual is associated with the presence and or amount of mutant nucleic acid sequence. - View Dependent Claims (44)
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45. A method for drug sensitivity of an individual diagnosed with cancer, said method comprising:
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a) obtaining a sample comprising nucleic acid from the individual; b) fragmenting the sample nucleic acid 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) amplifying the mutant nucleic acid with a pair of primers, wherein a first primer is specific for the mutant nucleic acid; d) detecting the presence, absence and/or amount of an amplification product containing the mutant sequence; and e) relating the presence, absence and/or amount of an amplification product containing the mutant sequence to cancer drug sensitivity.
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Specification