Thermostable ligase mediated DNA amplification system for the detection of genetic diseases
First Claim
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1. A method for distinguishing a first nucleotide sequence which differs by at least a single base from a second nucleotide sequence comprising:
- providing a sample potentially containing the first nucleotide sequence and the second nucleotide sequence;
providing a first oligonucleotide set of at least two oligonucleotides suitable for ligation together at a first ligation junction and for hybridization without mismatch at the first ligation junction to the first nucleotide sequence but not to the second nucleotide sequence, wherein the at least two oligonucleotides hybridize adjacent to one another on the first nucleotide sequence and have a hybridization temperature of about 50°
C. to 85°
C.;
providing a thermocyclable ligase which does not become irreversibly denatured and lose its catalytic activity when subjected to temperatures ranging from about 50°
C. to 105°
C.;
blending the sample, the at least two oligonucleotides, and the thermocyclable ligase to form an amplification mixture;
subjecting the amplification mixture to a series of cycles comprising a denaturation treatment, wherein the hybridized first oligonucleotide set is separated from the first nucleotide sequence or from the second nucleotide sequence, and a thermal hybridization treatment at a temperature of 50°
-85°
C., wherein the oligonucleotides of the first oligonucleotide set, when hybridized to the first nucleotide sequence, ligate to one another to amplify linearly a sequence of nucleotides complementary to the first nucleotide sequence and, when hybridized to the second nucleotide sequence, do not ligate together and individually separate from the second nucleotide sequence during the denaturation treatment; and
detecting the presence of the first nucleotide sequence in the sample by detecting the presence of ligated oligonucleotides of the first oligonucleotide set.
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Abstract
The present invention relates to the cloning of the gene of a thermophilic DNA ligase, from Thermus aquaticus strain HB8, and the use of this ligase for the detection of specific sequences of nucleotides in a variety of nucleic acid samples, and more particularly in those samples containing a DNA sequence characterized by a difference in the nucleic acid sequence from a standard sequence including single nucleic acid base pair changes, deletions, insertions or translocations.
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Citations
89 Claims
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1. A method for distinguishing a first nucleotide sequence which differs by at least a single base from a second nucleotide sequence comprising:
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providing a sample potentially containing the first nucleotide sequence and the second nucleotide sequence; providing a first oligonucleotide set of at least two oligonucleotides suitable for ligation together at a first ligation junction and for hybridization without mismatch at the first ligation junction to the first nucleotide sequence but not to the second nucleotide sequence, wherein the at least two oligonucleotides hybridize adjacent to one another on the first nucleotide sequence and have a hybridization temperature of about 50°
C. to 85°
C.;providing a thermocyclable ligase which does not become irreversibly denatured and lose its catalytic activity when subjected to temperatures ranging from about 50°
C. to 105°
C.;blending the sample, the at least two oligonucleotides, and the thermocyclable ligase to form an amplification mixture; subjecting the amplification mixture to a series of cycles comprising a denaturation treatment, wherein the hybridized first oligonucleotide set is separated from the first nucleotide sequence or from the second nucleotide sequence, and a thermal hybridization treatment at a temperature of 50°
-85°
C., wherein the oligonucleotides of the first oligonucleotide set, when hybridized to the first nucleotide sequence, ligate to one another to amplify linearly a sequence of nucleotides complementary to the first nucleotide sequence and, when hybridized to the second nucleotide sequence, do not ligate together and individually separate from the second nucleotide sequence during the denaturation treatment; anddetecting the presence of the first nucleotide sequence in the sample by detecting the presence of ligated oligonucleotides of the first oligonucleotide set. - 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, 41, 42, 43, 44)
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45. A method for together amplifying and distinguishing nucleotide sequences complementary to a first nucleotide sequence and a second nucleotide sequence, wherein the first and second nucleotide sequences differ by at least one base, comprising:
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providing a sample potentially containing the first nucleotide sequence and the second nucleotide sequence; providing a first oligonucleotide set of at least two oligonucleotides suitable for ligation together at a first ligation junction and for hybridization without mismatch at the first ligation junction to the first nucleotide sequence, but not to the second nucleotide sequence, wherein the oligonucleotides of the first set hybridize adjacent to one another on the first nucleotide sequence and have a hybridization temperature of about 50°
C. to 85°
C.;providing a second oligonucleotide set of at least two oligonucleotides suitable for ligation together at a second ligation junction and for hybridization without mismatch at the second ligation junction to the second nucleotide sequence, but not to the first nucleotide sequence, wherein the oligonucleotides of the second set hybridize adjacent to one another on the second nucleotide sequence and have a hybridization temperature of about 50°
C. to 85°
C.;providing a thermocyclable ligase which does not become irreversibly denatured and lose its catalytic activity when subjected to temperatures ranging from about 50°
C. to 105°
C.;blending the sample, the first set of oligonucleotides, and the thermocyclable ligase to form a first amplification mixture; blending the sample, the second set of oligonucleotides, and the thermocyclable ligase to form a second amplification mixture; subjecting the first and second amplification mixtures to a series of cycles comprising a denaturation treatment, wherein the first oligonucleotide set is separated from the first nucleotide sequence while the second oligonucleotide set is separated from the second nucleotide sequence, and a thermal hybridization treatment at a temperature of about 50°
C. to 85°
C., wherein the first oligonucleotide set hybridizes to the first nucleotide sequence and its oligonucleotides ligate to one another while the second oligonucleotide set hybridizes to the second nucleotide sequence and its oligonucleotides ligate to one another, to amplify linearly nucleotide sequences complementary to the first nucleotide sequence and to the second nucleotide sequence;detecting the presence of the first nucleotide sequence in the sample by detecting the presence of ligated oligonucleotides of the first oligonucleotide set; and detecting the presence of the second nucleotide sequence in the sample by detecting the presence of ligated oligonucleotides of the second oligonucleotide set. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89)
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Specification