Method for producing DNA from mRNA
US5851805A · kind A · utility
Assignee
Inventors
Key dates
| Filing date | Jan 16, 1997 |
| Grant date | Dec 22, 1998 |
| Priority date | — |
| Expiry date | Jan 16, 2017 |
Classification
- Technology area (CPC Y)Emerging Cross-Sectional Technologies
- CPC primaryY10S435/81
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
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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