Construction of uni-directionally cloned cDNA libraries from messenger RNA for improved 3′ end DNA sequencing

The present invention relates generally to the field of amplifying nucleic acids, more particularly to methods for producing cDNA from mRNA, sequencing DNA, and constructing cDNA libraries. Methods are provide for preparing cDNA corresponding to a mRNA. In the subject methods, a mRNA is first contac...

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Bibliographic Details
Main Authors Fu, Glenn K, Starnes, Steven, Stuve, Laura L
Format Patent
LanguageEnglish
Published 14.05.2002
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Summary:The present invention relates generally to the field of amplifying nucleic acids, more particularly to methods for producing cDNA from mRNA, sequencing DNA, and constructing cDNA libraries. 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.