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    • 7. 发明申请
    • DETECTION AND CONFIRMATION OF NUCLEIC ACID SEQUENCES BY USE OF OLIGONUCLEOTIDES COMPRISING A SUBSEQUENCE HYBRIDIZING EXACTLY TO A KNOWN TERMINAL SEQUENCE AND A SUBSEQUENCE HYBRIDIZING TO AN UNIDENTIFIED SEQUENCE
    • 核苷酸序列的检测和确认通过使用包含随机杂交的寡核苷酸完全杂交到已知的终末序列和随机杂交到未经鉴别的序列
    • WO9907896A3
    • 1999-04-29
    • PCT/US9816548
    • 1998-08-07
    • CURAGEN CORPROTHBERG JONATHAN MDEEM MICHAEL WSIMPSON JOHN W
    • ROTHBERG JONATHAN MDEEM MICHAEL WSIMPSON JOHN W
    • C12N15/09C12N15/10C12Q1/68
    • C12N15/1093C12Q1/6809C12Q1/6813C12Q1/6855C12Q2525/191C12Q2565/125
    • The present invention discloses a methodology which is directed to providing positive confirmation that nucleic acids, possessing putatively identified sequences predicted to generate observed GeneCalling signals, are actually present within the sample from which the signal was originally derived. The putatively identified nucleic acid fragment within the sample possesses 3'- and 5'-ends with known terminal subsequences, said method comprising: contacting said nucleic acid fragments in said sample in amplifying conditions with (i) a nucleic acid polymerase; (ii) "regular" primer oligonucleotides having sequences comprising hybridizable portions of said known terminal subsequences; and (iii) a "poisoning" oligonucleotide primer, said poisoning primer having a sequence comprising a first subsequence that is a portion of the sequence of one of said known terminal subsequences and a second subsequence that is a hybridizable portion of said putatively unidentified sequence which is adjacent to said one known terminal subsequence, wherein nucleic acids amplified with said poisoning primer are distinguishable upon detection from nucleic acids amplified with said nucleic acids amplified only with said regular primers; separating the products of the contacting step; and the detecting sequence is confirmed if the nucleic acids amplified with said poisoning primer are detected.
    • 本发明公开了一种旨在提供肯定确认的方法,核酸具有预测产生观察到的GeneCalling TM信号的推定鉴定序列实际上存在于最初得到信号的样本内。 所述样品中的推测鉴定的核酸片段具有已知末端亚序列的3'和5'末端,所述方法包括:使扩增条件下的所述样品中的所述核酸片段与(i)核酸聚合酶; (ii)具有包含所述已知末端子序列的可杂交部分的序列的“规则”引物寡核苷酸; 所述中毒引物具有包含作为所述已知末端子序列之一序列的一部分的第一子序列和作为所述假定未鉴定序列的可杂交部分的第二子序列的序列, 与所述一个已知末端子序列相邻,其中用所述中毒引物扩增的核酸在从用所述常规引物扩增的所述核酸扩增的核酸检测时是可区分的; 分离接触步骤的产物; 如果检测到用所述中毒引物扩增的核酸,则确认检测序列。