Method for analyzing a nucleotide sequence
First Claim
1. A method for analyzing a target nucleotide sequence having a first segment, a second segment and a third segment therebetween which third segment is formed of at least one nucleotide but which is formed of less than four different nucleotides wherein the third segment has a nucleotide or nucleotide sequence in a first state or a different second state, said method comprising:
- a) providing a first polynucleotide which is immobilized on a solid support and which is at least partially complementary to the first segment of the target nucleotide sequence;
b) hybridizing the first polynucleotide to the first segment of the target nucleotide sequence under conditions suitable for hybridization;
c) providing a second polynucleotide which is at least partially complementary to the second segment of the target nucleotide sequence;
d) hybridizing the second polynucleotide to the second segment of the target nucleotide sequence under conditions suitable for hybridization wherein a gap of at least one nucleotide is present between one end of the first polynucleotide and one end of the second polynucleotide;
e) providing at least one nucleotide selected such that all the nucleotides which are complementary to the third segment are provided for only one of the first state or the second state and less than all the nucleotides complementary to the third segment are provided for the other state, the nucleotide or nucleotides being provided under conditions whereby the nucleotide or nucleotides form an extended portion of one of the first or second polynucleotide that is complementary to the nucleotides(s) of the third segment of the target nucleic acid sequence in one of the states;
f) linking the polynucleotide with the extended portion to the other polynucleotide under conditions whereby a fused product is formed comprised of the first polynucleotide, the second polynucleotide and the extended portion therebetween;
g) separating the fused product from the target nucleotide sequence and any non-linked second polynucleotide by treatment with exonuclease to digest target nucleotide sequence and non-linked second polynucleotide, and washing;
h) amplifying at least a portion of the fused product wherein the portion contains the extended portion under conditions suitable to form an amplified product containing the first polynucleotide and a detectable label, the amplifying being conducted to obtain a sufficient quantity of amplified product to detect the detectable label; and
i) detecting the presence of the label of the amplified product.
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Abstract
A method for analyzing a target nucleotide sequence which exists in a first state or a different second state which makes the method particularly useful for determining point mutations. The method uses a first polynucleotide which is immobilized on a solid support and which is at least partially complementary to a first segment of the target nucleotide sequence. By means of a series of steps, a product of the first polynucleotide and a further polynucleotide that contains a detectable label can be obtained. When the state to be analyzed occurs in a rare population, amplification can be conducted so that substantially only amplification of the target nucleotide sequence in one of the states is attained. The method can be used to analyze multiple target sequences simultaneously. A kit which can be used in the method is also set forth.
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Citations
38 Claims
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1. A method for analyzing a target nucleotide sequence having a first segment, a second segment and a third segment therebetween which third segment is formed of at least one nucleotide but which is formed of less than four different nucleotides wherein the third segment has a nucleotide or nucleotide sequence in a first state or a different second state, said method comprising:
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a) providing a first polynucleotide which is immobilized on a solid support and which is at least partially complementary to the first segment of the target nucleotide sequence; b) hybridizing the first polynucleotide to the first segment of the target nucleotide sequence under conditions suitable for hybridization; c) providing a second polynucleotide which is at least partially complementary to the second segment of the target nucleotide sequence; d) hybridizing the second polynucleotide to the second segment of the target nucleotide sequence under conditions suitable for hybridization wherein a gap of at least one nucleotide is present between one end of the first polynucleotide and one end of the second polynucleotide; e) providing at least one nucleotide selected such that all the nucleotides which are complementary to the third segment are provided for only one of the first state or the second state and less than all the nucleotides complementary to the third segment are provided for the other state, the nucleotide or nucleotides being provided under conditions whereby the nucleotide or nucleotides form an extended portion of one of the first or second polynucleotide that is complementary to the nucleotides(s) of the third segment of the target nucleic acid sequence in one of the states; f) linking the polynucleotide with the extended portion to the other polynucleotide under conditions whereby a fused product is formed comprised of the first polynucleotide, the second polynucleotide and the extended portion therebetween; g) separating the fused product from the target nucleotide sequence and any non-linked second polynucleotide by treatment with exonuclease to digest target nucleotide sequence and non-linked second polynucleotide, and washing; h) amplifying at least a portion of the fused product wherein the portion contains the extended portion under conditions suitable to form an amplified product containing the first polynucleotide and a detectable label, the amplifying being conducted to obtain a sufficient quantity of amplified product to detect the detectable label; and i) detecting the presence of the label of the amplified product. - 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)
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27. A method for analyzing a target nucleotide sequence having a first segment, a second segment and a third segment therebetween which third segment is formed of at least one nucleotide but which is formed of less than four different nucleotides wherein the third segment has a nucleotide or nucleotide sequence in a first state or a different second state, said method comprising:
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a) providing a first polynucleotide which is immobilized on a solid support and which is at least partially complementary to the first segment of the target nucleotide sequence; b) providing a second polynucleotide which is at least partially complementary to the second segment of the target nucleotide sequence; c) hybridizing the second polynucleotide to the second segment of the target nucleotide sequence under conditions suitable for hybridization; d) providing at least one nucleotide selected such that all the nucleotides which are complementary to the third segment are provided for only one of the first state or the second state and less than all the nucleotides complementary to the third segment are provided for the other state, the nucleotide or nucleotides being provided under conditions whereby the nucleotide or nucleotides form an extended portion of the second polynucleotide that is complementary to the nucleotides(s) of the third segment of the target nucleic acid sequence in one of the states; e) hybridizing the first polynucleotide to the first segment of the target nucleotide sequence under conditions suitable for hybridization; f) linking the polynucleotide with the extended portion to the other polynucleotide under conditions whereby a fused product is formed comprised of the first polynucleotide, the second polynucleotide and the extended portion therebetween; g) separating the fused product from the target nucleotide sequence and any non-linked second polynucleotide by treatment with exonuclease to digest target nucleotide sequence and non-linked second polynucleotide, and washing; h) amplifying at least a portion of the fused product wherein the portion contains the extended portion under conditions suitable to form an amplified product containing the first polynucleotide and a detectable label, the amplifying being conducted to obtain a sufficient quantity of amplified product to detect the detectable label; and i) detecting the presence of the label of the amplified product. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36, 37)
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38. A method for analyzing a target nucleotide sequence having a first segment, a second segment and a third segment therebetween which third segment is formed of at least one nucleotide but which is formed of less than four different nucleotides wherein the third segment has a nucleotide or nucleotide sequence in a first state or a different second state, said method comprising:
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a) providing a first polynucleotide which is immobilized on a solid support and which is at least partially complementary to the first segment of the target nucleotide sequence; b) hybridizing the first polynucleotide to the first segment of the target nucleotide sequence under conditions suitable for hybridization; c) providing a second polynucleotide which is at least partially complementary to the second segment of the target nucleotide sequence; d) hybridizing the second polynucleotide to the second segment of the target nucleotide sequence under conditions suitable for hybridization wherein a gap of at least one nucleotide is present between one end of the first polynucleotide and one end of the second polynucleotide, and wherein the polynucleotide having a 3'"'"' end adjacent the gap is modified so that ligation across the gap is inhibited; e) providing at least one nucleotide selected such that all the nucleotides which are complementary to the third segment are provided for only one of the first state or the second state and less than all the nucleotides complementary to the third segment are provided for the other state, the nucleotide or nucleotides being provided under conditions whereby the nucleotide or nucleotides form an extended portion of one of the first or second polynucleotide that is complementary to the nucleotides(s) of the third segment of the target nucleic acid sequence in one of the states; f) linking the polynucleotide with the extended portion to the other polynucleotide under conditions whereby a fused product is formed comprised of the first polynucleotide, the second polynucleotide and the extended portion therebetween; g) separating the fused product from the target nucleotide sequence and any non-linked second polynucleotide; h) amplifying at least a portion of the fused product wherein the portion contains the extended portion under conditions suitable to form an amplified product containing the first polynucleotide and a detectable label, the amplifying being conducted to obtain a sufficient quantity of amplified product to detect the detectable label; and i) detecting the presence of the label of the amplified product.
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Specification