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Construction of uni-directionally cloned cDNA libraries from messenger RNA for improved 3amp;prime; end DNA sequencing
Construction of uni-directionally cloned cDNA libraries from messenger RNA for improved 3amp;prime; end DNA sequencing
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机译:从信使RNA单向克隆cDNA文库的构建以提高3-和prime;末端DNA测序
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摘要
Methods are provide for preparing cDNA corresponding to a mRNA. In the subject methods, a mRNA is first contacted with a mixture of primers under first strand cDNA synthesis conditions. The primer mixture contains primers that have at least 10 contiguous deoxythymidines, a double stranded restriction enzyme recognition sequence near one end and a non-polyA-complementary region near the other end, where the non-polyA-complementary region is -VV, -VTV, -VTTV, -VTTTV, and -VVVVV. The resultant cDNA is modified such that the polyT tail is substantially removed. The modified cDNA is then ligated into a vector. The subject methods find use in a variety of applications, and find particular use in the sequencing of DNA and in the synthesis of cDNA libraries.
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