Method for producing DNA from mRNA

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical

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536 221, 536 242, 536 2453, 536 2534, 536 254, 435 9152, 435395, 435810, 435 71, 435 9151, 424 858, C07H 1900, C07H 2104, C07H 2100, C12P 1934

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058518053

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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