Determination of nucleotide sequence variations through limited primer extension
First Claim
1. A method of analyzing a variant site of a target nucleic acid, comprising(a) conducting a template-dependent extension reaction comprising extending a primer in the presence of the target nucleic acid and a mixture of nucleotides comprising at least one labeled extendible nucleotide and at least one labeled non-extendible nucleotide, each labeled extendible nucleotide and labeled non-extendible nucleotide being complementary to a different allelic form of the target nucleic acid and optionally differentially labeled, wherein the primer hybridizes to a segment of the target nucleic acid such that the 3′
- -end of the primer hybridizes adjacent the variant site of the target nucleic acid, whereby if the labeled extendible nuclcotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of the labeled extendible nucleotide, and can be extended further if one or more nucleotides downstream of the variant site are complementary to one of the nucleotides in the mixture, and if the labeled non-extendible nucleotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of the labeled non-extendible nucleotide; and
(b) detecting incorporation of labeled nucleotide into the extended primer, the identity of the labeled nucleotide incorporated into the primer indicating the identity of the nucleotide at the variant site, wherein the identity of the incorporated nucleotide is determined from the label borne by the incorporated nucleotide and/or the size of the extended primer.
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Abstract
The present invention provides methods and kits for determining the identity of a nucleotide at a variant site in a target nucleic acid of interest, including, for example, point mutations and single nucleotide polymorphisms. The methods involve conducting template-dependent extension reactions in the presence of a mixture of nucleotides that include at least one labeled extendible nucleotide and at least one labeled non-extendible nucleotide that are selected to be complementary to the nucleotides that potentially occupy the variant site. The particular labeled nucleotide incorporated into the extension products is characteristic of the nucleotide at the variant site. In addition to their utility in analyzing point mutations and single nucleotide polymorphisms, the methods and kits of the invention have utility in a variety of other applications in which specific nucleotide sequence information is of value, including, for example, paternity disputes, prenatal testing, forensic analysis and detection of pathogens.
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Citations
45 Claims
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1. A method of analyzing a variant site of a target nucleic acid, comprising
(a) conducting a template-dependent extension reaction comprising extending a primer in the presence of the target nucleic acid and a mixture of nucleotides comprising at least one labeled extendible nucleotide and at least one labeled non-extendible nucleotide, each labeled extendible nucleotide and labeled non-extendible nucleotide being complementary to a different allelic form of the target nucleic acid and optionally differentially labeled, wherein the primer hybridizes to a segment of the target nucleic acid such that the 3′ - -end of the primer hybridizes adjacent the variant site of the target nucleic acid, whereby
if the labeled extendible nuclcotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of the labeled extendible nucleotide, and can be extended further if one or more nucleotides downstream of the variant site are complementary to one of the nucleotides in the mixture, and if the labeled non-extendible nucleotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of the labeled non-extendible nucleotide; and
(b) detecting incorporation of labeled nucleotide into the extended primer, the identity of the labeled nucleotide incorporated into the primer indicating the identity of the nucleotide at the variant site, wherein the identity of the incorporated nucleotide is determined from the label borne by the incorporated nucleotide and/or the size of the extended 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, 42)
(a) multiple copies of the primer are provided and the target nucleic acid is a first and/or second target nucleic acid contained in a sample from a diploid subject, the first and/or second target nucleic acid differing at the site of variation, whereby if the first target nucleic acid is present, at least one copy of the primer is extended by incorporation of the labeled deoxynucleotide and if the second target nucleic acid is present, at least one copy of the primer is extended by incorporation of the labeled non-extendible nucleotide; - and
(b) detecting comprises detecting incorporation of labeled deoxynucleotide and/or labeled non-extenidiblc nticleotide into the primer copies, incorporation of only labeled deoxynuclCotide or labeled non-extendible nucleotide indicating that the subject is a homozygote, whereas incorporation of labeled deoxynucleotide and labeled non-extenidible nucleotide indicate that the subject is a heterozygote.
- -end of the primer hybridizes adjacent the variant site of the target nucleic acid, whereby
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26. The method of claim 25, wherein the labeled deoxynucleotide and labeled non-extendible nucleotide bear different fluorescent labels that emit at different wavelengths, and detection comprises detection of fluorescence, detection of fluorescence at a single wavelength indicating that the subject is a homozygote and detection of fluorescence at two wavelengths indicating that the subject is a heterozygote.
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27. The method of claim 25, wherein:
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(a) one or more nuclCotidcs immediately downstream of the variant site of the target nucleic acids are the same as a nuLcleotidc potentially at the variant site and the labeled deoxynucleotidc is selected to be complementary to the one or more downstream nucleotides, whereby if the labeled deoxynucleotidc is complementary to the nucleotide at the variant site of either the first or second target nucleic acid, then the primer is further extended to include additional labeled deoxynuLcleotide such that the first and second target nucleic acid gencerate different sized extension products; and
(b) detecting comprises detecting the presence or absence of different sized extension products.
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42. The method of claim 1, wherein detection of incorporated labeled nucleotide is performed without separating labeled nucleotides from extended primer.
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28. A method for analyzing variant sites in one or more target nucleic acids, comprising:
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(a) conducting a plurality of template-dependent extension reactions in the presence of a plurality of different primers, wherein different primers hybridize adjacent to different variant sites of the one or more target nucleic acids and are differentially labeled, each extension reaction comprising (i) contacting a sample containing the target nucleic acid(s) with one of the different labeled primers, wherein the 3′
-end of the primer hybridizes adjacent to but not including the variant site of one of the target nucleic acids, and(ii) exposing the primer to a mixture of nucleotides comprising at least one labeled extendible nuclcotidc and at least one labeled non-extendible nucleotide under conditions whereby if the labeled extendible nucleotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of labeled extendible nucleotide, and can be extended further if one or more nucleotides downstream of the variant site are complementary to one of the nucleotides in the mixture, and if the labeled non-extendible nucleotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of labeled non-extendible nucleotide, whereby the extension reactions generate a plurality of different extension products, extension products generated from different variant sites being distinguishable on the basis of the different labels borne by the extended primers; and
(b) detecting the incorporation of labeled nucleotides into the extension products as an indication of the nucleotides occupying the site of variation in the target nucleic acids, wherein the identity of the incorporated nucleotide is determined from the label borne by the incorporated nucleotide and/or the size of the extended primer. - View Dependent Claims (29, 30, 31, 32, 33)
(a) the different labels borne by the different primers are different fluorophores and the labeled nucleotides bear different fluorophores, such that the fluorophores borne by an extension product form a donor and acceptor pair; - and
(b) detecting comprises detecting a change in an emission from the donor and/or acceptor for the different donor and acceptor pairs.
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33. The method of claim 28, wherein detection is performed by fluorescence polarization.
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34. A kit for analyzing a variant site in a target nucleic acid, comprising:
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(a) at least one labeled extendible nucleotide and at least one labeled non-extendible nucleotide, the nucleotides complementary to different allelic forms of the target nucleic acid; and
(b) at least one primer, each primer hybridizing to a segment of the target nucleic acid such that the 3′
end of the primer is adjacent the variant site of the target nucleic acid.- View Dependent Claims (35, 36, 37, 38, 39, 40, 41)
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43. A method of analyzing a variant site of a target nucleic acid, comprising:
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(a) conducting a template-dependent extension reaction comprising extending a primer in the presence of the target nucleic acid and a mixture of nucleotides comprising at least one labeled extendible nucleotide and at least one labeled non-extendible nucleotide, each labeled extendible nucleotide and labeled non-extendible nucleotide being complementary to a different allelic form of the target nucleic acid and differentially labeled, and wherein the primer hybridizes to a segment of the target nucleic acid such that the 3′
-end of the primer hybridizes adjacent the variant site of the target nucleic acid, wherebyif the labeled extendible nucleotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of the labeled extendible nucleotide, and if the labeled non-extendible nucleotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of the labeled non-extendible nucleotide; and
(b) detecting incorporation of labeled nucleotide into the extended primer, the identity of the labeled nucleotide incorporated into the primer indicating the identity of the nucleotide at the variant site, wherein the identity of the incorporated nucleotide is determined from the label borne by the incorporated nucleotide. - View Dependent Claims (44, 45)
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Specification