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Genome Res. 2001 Sep;11(9):1553-8.

Construction of long-transcript enriched cDNA libraries from submicrogram amounts of total RNAs by a universal PCR amplification method.

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1
Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.

Abstract

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 approximately 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.

PMID:
11544199
PMCID:
PMC311119
DOI:
10.1101/gr.185501
[Indexed for MEDLINE]
Free PMC Article

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