Analysis of nucleotide polymorphisms at a site
First Claim
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1. A method for determining the identity of a polymorphic nucleotide in a target sequence having at least two known variant nucleotides at a site, comprising:
- performing a primer extension reaction with the target sequence using an extension reaction mixture comprising;
a primer that specifically hybridizes to the target sequence such that there is a one or more nucleotide gap between the 3′
terminus of the primer and the variant nucleotide of the polymorphic site of the target sequence, and a mixture of deoxyribonucleoside triphosphates (dNTPs) or ribonucleoside triphosphates (rNTPs), where the mixture of dNTPs or rNTPs provide for at least one nucleotide extension of the primer when hybridized to a target sequence having either of the two variant nucleotides at the polymorphic site, wherein the mixture excludes a dNTP or rNTP complementary to one of said variant nucleotides of the polymorphic site, and wherein the dNTPs or rNTPs in the mixture are not detectably labeled or modified, and wherein the extension reaction is performed in the absence of a dideoxynucleoside triphosphate (ddNTP); and
analyzing primer extension products of said extension reaction;
wherein the length of the primer extension products is indicative of the identity of the variant nucleotides at the polymorphic site.
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Abstract
The identity of the polymorphic nucleotide in a target sequence having at least two known variants can be easily and efficiently detected by hybridizing at least one primer upstream of the biallelic marker and performing extension reactions using the target DNA with the hybridized primer, where a first reaction is conducted in the absence of a deoxyribonucleoside triphosphate or ribonucleoside triphosphate complementary to the first known variant, and a second reaction is conducted in absence of a deoxyribonucleoside triphosphate or ribonucleoside triphosphate complementary to the second known variant. Determining the lengths of the primers and any extension products from both reactions will indicate which variant or variants are present in a DNA sample.
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Citations
19 Claims
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1. A method for determining the identity of a polymorphic nucleotide in a target sequence having at least two known variant nucleotides at a site, comprising:
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performing a primer extension reaction with the target sequence using an extension reaction mixture comprising;
a primer that specifically hybridizes to the target sequence such that there is a one or more nucleotide gap between the 3′
terminus of the primer and the variant nucleotide of the polymorphic site of the target sequence, anda mixture of deoxyribonucleoside triphosphates (dNTPs) or ribonucleoside triphosphates (rNTPs), where the mixture of dNTPs or rNTPs provide for at least one nucleotide extension of the primer when hybridized to a target sequence having either of the two variant nucleotides at the polymorphic site, wherein the mixture excludes a dNTP or rNTP complementary to one of said variant nucleotides of the polymorphic site, and wherein the dNTPs or rNTPs in the mixture are not detectably labeled or modified, and wherein the extension reaction is performed in the absence of a dideoxynucleoside triphosphate (ddNTP); and
analyzing primer extension products of said extension reaction;
wherein the length of the primer extension products is indicative of the identity of the variant nucleotides at the polymorphic site. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method for screening a DNA sample for a plurality of target sequences having at least two known variants, comprising:
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contacting a sample comprising a plurality of known target sequences with an extension reaction mixture to produce primer extension reaction products, the extension reaction mixture comprising a primer that specifically hybridizes to a target sequence of interest such that there is one or more nucleotide gap between the 3′
terminus of the primer and one the variant nucleotide of the polymorphic site of the target sequence, anda mixture of deoxyribonucleoside triphosphates (dNTPs) or ribonucleoside triphosphates (rNTPs), where the dNTPs or rNTPs in the mixture provide for at least one nucleotide extension of the primer when hybridized to a target sequence having either of the two variant nucleotides, the mixture excluding a dideoxynucleoside triphosphate (ddNTP) and further excluding a dNTP or rNTP complementary to one of said variant nucleotides of the SNP, wherein the dNTPs or rNTPs in the mixture are not detectably labeled or modified; and
analyzing the primer extension products;
wherein the length of the primer extension products is indicative of the identity of the variant nucleotides at the polymorphic site. - View Dependent Claims (16, 17, 18, 19)
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Specification
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Current AssigneeTecholding S.A.
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Original AssigneeTetragen SA
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InventorsMerenkova, Irena N.
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Primary Examiner(s)SITTON, JEHANNE SOUAYA
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Application NumberUS09/471,703Time in Patent Office1,664 DaysField of Search435/6, 435/91.2, 536/23.1, 536/24.3, 536/24.33US Class Current435/6.11CPC Class CodesC12Q 1/6827 for detection of mutation o...C12Q 1/6858 Allele-specific amplificationC12Q 2535/125 Allele specific primer exte...
