Construction of uni-directionally cloned cDNA libraries from messenger RNA for improved 3′ end DNA sequencing
US6387624B1 · kind B1 · utility
Assignee
Inventors
Key dates
| Filing date | Apr 14, 2000 |
| Grant date | May 14, 2002 |
| Priority date | — |
| Expiry date | Apr 14, 2020 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N15/1096
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
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.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.