Construction of long-transcript enriched cDNA libraries from submicrogram amounts of total RNAs by a universal PCR amplification method.
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.
Piao
Y
Y
Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
Ko
N T
NT
Lim
M K
MK
Ko
M S
MS
D000818:Animals; D018076:DNA, Complementary / Q000235:genetics*; D005784:Gene Amplification; D015723:Gene Library; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis* / Q000235:genetics*
D018076:DNA, Complementary; D012313:RNA