Methods and compositions for producing 5' enriched cDNA librarie

Details Methods and compositions for producing 5' enriched cDNA librarie Methods and compositions for producing 5' enriched cDNA librarie

1999-06-30

2000-07-04

Elliott, George C.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 911, 435 6, 536 231, 536 243, C07H 2102, C07H 2104, C12P 1934, C12Q 168

Patent

active

060837276

ABSTRACT:
Methods are provided for preparing a double-stranded cDNA corresponding to the 5' end of an mRNA. In the subject methods, an mRNA is first contacted with an oligo dT primer under first strand cDNA synthesis conditions. Next, the resultant hybrid is contacted with a random primer under first strand cDNA synthesis conditions, such that a cDNA complementary to the 5' end of the mRNA is produced. The resultant hybrid molecule is converted to two different double-stranded cDNAs, a first cDNA comprising the oligo dT primer and a second cDNA lacking the oligo dT primer. The two double-stranded cDNAs are then separated from each other. The subject methods find use in a variety of applications, and find particular use in the synthesis of 5' enriched cDNA libraries. Also provided are cDNA libraries produced by the subject methods, as well as kits for performing the subject methods.

REFERENCES:
Promega "Protocols & Applications Guide" 1991.
Pharmacia Biotech, 1996 Catalog p. 94-103.
Dear et al Mammalian Genome 7, 654-656 (1996).
Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 1989) Chapter 8, pp. 8.2-8.86.

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