发明名称 |
Methods and compositions for generating full-length cDNA having arbitrary nucleotide sequence at the 3'-end |
摘要 |
Described are compositions and methods which allow for the efficient addition of a defined sequence at the 3'-end of a full-length cDNA in the course of first-strand cDNA synthesis from an mRNA template. A cDNA synthesis primer that is capable of annealing to mRNA is used to prime the first strand synthesis reaction. An oligonucleotide that is linked to the 5'-end of the mRNA serves as a short, extended template such that when the reverse transcriptase enzyme reaches the 5'-end of the mRNA, the enzyme switches templates and proceeds to transcribe through the end of the linked oligonucleotide. As a result, the single-stranded cDNA product which corresponds to the full-length mRNA, will have at the 3'-end a defined sequence which is complementary to the linked oligonucleotide. A conservative element in the oligonucleotide sequence responsible for this reaction can include 3 to 5 guanylic acid residues at the 3'-end of the oligonucleotide. The subject invention provides for the increased synthesis of full-length cDNA from mRNA templates. The full-length cDNA prepared according to the present invention can then be amplified using PCR or cloned using standard procedures.
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申请公布号 |
US5962271(A) |
申请公布日期 |
1999.10.05 |
申请号 |
US19960582562 |
申请日期 |
1996.01.03 |
申请人 |
CLOUTECH LABORATORIES, INC. |
发明人 |
CHENCHIK, ALEX;ZHU, YORK;DIATCHENKO, LUDA;SIEBERT, PAUL |
分类号 |
C12N15/09;C12N15/10;C12Q1/68;(IPC1-7):C12P19/34;C07H21/02;C07H21/04 |
主分类号 |
C12N15/09 |
代理机构 |
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主权项 |
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地址 |
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