Construction of Long-Transcript Enriched cDNA Libraries from Submicrogram Amounts of Total RNAs by a Universal PCR Amplification Method
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 ∼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 numbersBG060207–BG062928.]
Footnotes
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↵1 Corresponding author.
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E-MAIL kom{at}grc.nia.nih.gov; FAX (410) 558-8331.
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Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.185501.
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- Received February 21, 2001.
- Accepted June 4, 2001.
- Cold Spring Harbor Laboratory Press
