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

Genome research ·Vol. 11 ·No. 9 ·2001-09-00 ·Pages 1553-8

Piao Y, Ko NT, Lim MK, Ko MS

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.

MeSH Terms

Animals DNA, Complementary/genetics Gene Amplification Gene Library Mice Mice, Inbred C57BL Molecular Sequence Data Polymerase Chain Reaction/methods RNA/analysis,genetics

Chemicals

DNA, Complementary RNA

Authors & Affiliations

4 authors, click to expand affiliations / ORCID

Piao Y

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

Ko N T

Lim M K

Ko M S

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Article Info

Journal

Genome research

Abbr.

Genome Res

ISSN

1088-9051

DOI

10.1101/gr.185501

Published

2001-09-00

Pages

1553-8

Language

English

Region

United States

NLM ID

9518021

PMCID

PMC311119

Subset

Databases

GENBANK

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PubMed source DOI: 10.1101/gr.185501 PMC Full Text View journal details

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

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