Detection of sequence variation of nucleic acid by shifted termination analysis
First Claim
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1. A method for detecting or quantifying a target nucleic acid in a sample comprising:
- (a) preparing a primer complementary to a sequence immediately upstream of a target nucleotide base at a predetermined position in a template of a nucleic acid of interest;
(b) treating a sample containing the nucleic acid of interest, if the nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases spanning the specific position, or directly employing step (c) if the nucleic acid of interest is single-stranded;
(c) annealing the primer from (a) with the target nucleic acid from (b) under high stringency conditions to obtain a primer-nucleic acid duplex, wherein the target nucleotide base in the nucleic acid of interest is the first unpaired base immediately downstream of the 3′
end of the primer;
(d) mixing the primer-nucleic acid duplex from (c) with a primer extension reaction reagent comprising;
(i) one type of terminator nucleotide or optionally, absence of a nucleotide, that is complementary to the target base at the predetermined position of the nucleic acid of interest, and (ii) three types of non-terminator nucleotides that are different from the terminator nucleotide in (i), and at least one type is optionally labeled with a detectable marker;
. (e) performing the primer extension reaction by enzymatic or chemical means, wherein the incorporation of said terminator nucleotide or non-terminator nucleotide to the primer depends upon the identity of the unpaired nucleotide base in the nucleic acid template immediately downstream of the 3′
end of the primer, and wherein incorporation of said terminator nucleotidec in the sequence complementary to said target nucleotide base in the nucleic acid of interest will terminate said primer extension without incorporating any labeled non-terminator nucleotide into the primer, wherein said primer is not labeled, and further wherein, when the target nucleotide base is changed to any other type of nucleotide, one of the non-terminator nucleotides labeled with said detectable maker, or optionally not labeled with any marker if mass spectrometry is used as a detecting method, that is complementary to the mutated nucleotide base, is sequence-dependently incorporated into the primer by said primer extension reaction; and
(f) determining the presence and identity of the nucleotide base at the predetermined position in the nucleic acid of interest by detecting the incorporated labeled non-terminator in the primer.
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Abstract
The invention relates to a method for detecting any mutation at a predetermined site occurring in a known nucleic acid sequence. The method uses primer extension analysis to detect the mutation. Unlabeled terminator is supplied along with labeled non-terminator in the primer extension reaction to detect whether the first nucleic acid base on the template strand that is directly opposite the nucleic acid base immediately 3′ to a primer is a mutant. In the primer extension reaction, the terminator is complementary to the wild-type base on the template strand that is directly opposite the nucleic acid base immediately 3′ to the primer. Non-terminators are the other nucleotides and are labeled. When the terminator is incorporated into the primer extension strand, primer extension reaction terminates. Incorporation of a labeled non-terminator in the primer extension strand indicates that a mutation has occurred at the predetermined nucleic acid base site.
24 Citations
36 Claims
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1. A method for detecting or quantifying a target nucleic acid in a sample comprising:
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(a) preparing a primer complementary to a sequence immediately upstream of a target nucleotide base at a predetermined position in a template of a nucleic acid of interest;
(b) treating a sample containing the nucleic acid of interest, if the nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases spanning the specific position, or directly employing step (c) if the nucleic acid of interest is single-stranded;
(c) annealing the primer from (a) with the target nucleic acid from (b) under high stringency conditions to obtain a primer-nucleic acid duplex, wherein the target nucleotide base in the nucleic acid of interest is the first unpaired base immediately downstream of the 3′
end of the primer;
(d) mixing the primer-nucleic acid duplex from (c) with a primer extension reaction reagent comprising;
(i) one type of terminator nucleotide or optionally, absence of a nucleotide, that is complementary to the target base at the predetermined position of the nucleic acid of interest, and (ii) three types of non-terminator nucleotides that are different from the terminator nucleotide in (i), and at least one type is optionally labeled with a detectable marker;
.(e) performing the primer extension reaction by enzymatic or chemical means, wherein the incorporation of said terminator nucleotide or non-terminator nucleotide to the primer depends upon the identity of the unpaired nucleotide base in the nucleic acid template immediately downstream of the 3′
end of the primer, and wherein incorporation of said terminator nucleotidec in the sequence complementary to said target nucleotide base in the nucleic acid of interest will terminate said primer extension without incorporating any labeled non-terminator nucleotide into the primer, wherein said primer is not labeled, and further wherein, when the target nucleotide base is changed to any other type of nucleotide, one of the non-terminator nucleotides labeled with said detectable maker, or optionally not labeled with any marker if mass spectrometry is used as a detecting method, that is complementary to the mutated nucleotide base, is sequence-dependently incorporated into the primer by said primer extension reaction; and
(f) determining the presence and identity of the nucleotide base at the predetermined position in the nucleic acid of interest by detecting the incorporated labeled non-terminator in the primer. - 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)
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Specification
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Current AssigneeShinkatsu Morisawa
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Original AssigneeShinkatsu Morisawa
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InventorsWang, Xiao Bing
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Application NumberUS11/284,453Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current435/6CPC Class CodesC12Q 1/6858 Allele-specific amplificationC12Q 2525/185 incorporating bases where t...
