发明名称 Primers with Modified Phosphate and Base in Allele-Specific PCR
摘要 The present invention includes a method of allele-specific amplification, utilizing an allele-specific oligonucleotide, at least partially complementary to more than one variant of the target sequence, but having at least one selective nucleotide complementary to only one variant of the target sequence and incorporating both a nucleotide with a base covalently modified at the exocyclic amino group and a modified phosphate.
申请公布号 US2014170650(A1) 申请公布日期 2014.06.19
申请号 US201314092133 申请日期 2013.11.27
申请人 Roche Molecular Systems, Inc. 发明人 Froehner Stefanie;Heindl Dieter;Kessler Dirk;Schoenbrunner Nancy;Tsan Alison
分类号 C12Q1/68 主分类号 C12Q1/68
代理机构 代理人
主权项 1. A method of allele-specific amplification of a variant of a target sequence, the target existing in the form of several variant sequences, the method comprising: (a) hybridizing a first oligonucleotide and a second oligonucleotide to at least one variant of the target sequence; wherein the first oligonucleotide is at least partially complementary to one or more variants of the target sequence, and the second oligonucleotide is at least partially complementary to one or more variants of the target sequence, and has at least one selective nucleotide complementary to only one variant of the target sequence; wherein said second oligonucleotide comprises both a nucleotide with a base covalently modified at the exocyclic amino group and a modified phosphate having a structure: wherein A and B represents a nucleotide chain, D is OH or CH3, and Acc is an electron acceptor or an electron acceptor substituted with a residue R wherein R is an organic substituent, wherein Acc is selected from the group consisting of CN, SO2—R′, in which R′ comprises at least one amino-substituted alkyl, an optionally substituted aryl or an optionally substituted heterocycle, and a six membered N+ heterocycle with at least one alkylated N-atom in ortho- or para-position, said heterocycle selected from the group consisting of pyridinium, pyrimidinium, and quinolinium; (b) providing conditions suitable for oligonucleotide extension by a nucleic acid polymerase; (c) extending said first oligonucleotide and said second oligonucleotide by said nucleic acid polymerase, wherein said nucleic acid polymerase is capable of extending said second oligonucleotide efficiently when said oligonucleotide is hybridized to a variant of the target sequence which is complementary to said at least one selective nucleotide, and substantially less efficiently when said second oligonucleotide is hybridized to a variant of the target sequence which is not complementary to said at least one selective nucleotide.
地址 Pleasanton CA US