发明授权
- 专利标题: Method for producing DNA from mRNA
- 专利标题(中): 从mRNA产生DNA的方法
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申请号: US784751申请日: 1997-01-16
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公开(公告)号: US5851805A公开(公告)日: 1998-12-22
- 发明人: Dong Wei , Veronica M. Maher , Joseph Justin McCormick
- 申请人: Dong Wei , Veronica M. Maher , Joseph Justin McCormick
- 申请人地址: MI East Lansing
- 专利权人: Board of Trustees operating Michigan State University
- 当前专利权人: Board of Trustees operating Michigan State University
- 当前专利权人地址: MI East Lansing
- 主分类号: C12N15/10
- IPC分类号: C12N15/10 ; C12Q1/68 ; C07H19/00 ; C07H21/00 ; C07H21/04 ; C12P19/34
摘要:
An improved method for amplifying cDNA fragments corresponding to the mRNA species expressed in cells is described. The method involves priming the mRNA with a first labeled synthetic oligodeoxynucleotide and then reverse transcribing the mRNA to produce a mRNA:cDNA hybrid; and then producing a double-stranded DNA from the hybrid. This DNA is digested with a restriction endonuclease and then a synthetic DNA linker, carrying a second label, is ligated at the cut to the DNA strand containing the 5' phosphate group. The DNA is then isolated with a first reagent which binds the second label. The DNA fragments of interest are then isolated using a second reagent which binds the first label. The isolated DNA fragments are amplified using polymerase chain reaction, and then separated on a sequencing gel. The method is particularly useful for comparing the mRNA species expressed in the cells of various samples.
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