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Vol. 11, Issue 9, 1553-1558, September 2001

METHODS
Construction of Long-Transcript Enriched cDNA Libraries from Submicrogram Amounts of Total RNAs by a Universal PCR Amplification Method

Yulan Piao, Naomi T. Ko, Meng K. Lim, and Minoru S.H. Ko1

Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA

Here we report a novel design of linker primer that allows one to differentially amplify long tracts (average 3.0 kb with size ranges of 1-7 kb) or short DNAs (average 1.5 kb with size ranges of 0.5-3 kb) from a complex mixture. The method allows one to generate cDNA libraries enriched for long transcripts without size selection of insert DNAs. One representative library from newborn kidney includes 70% of clones bearing ATG start codons. A comparable library has been generated from 20 mouse blastocysts, containing only ~40 ng of total RNA. This universal PCR amplification scheme can provide a route to isolate very large cDNAs, even if they are expressed at very low levels.

[The sequence data described in this paper have been submitted to the GenBank data library under accession numbers BG060207-BG062928.]

1 Corresponding author.

11:1553-1558 ©2001 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/01 $5.00
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