International Mammalian Genome Society

The 16th International Mouse Genome Conference (2002)


POSTER 78 - CONSTRUCTION AND EVALUATION OF LONG-INSERT cDNA LIBRARIES FROM SMALL AMOUNTS OF MATERIALS

Y Piao
Developmental Genomics & Aging Section, Laboratory of Genetics; National Institute on Aging, NIH

Martin P, Bassey U, Stagg C, 1)Ko M
NIA

In order to recover long-insert cDNA clones for genes expressed only at limited times and places during embryogenesis, we have developed a method to construct a cDNA library from small numbers of cells (600-800cells). The method allows us to generate cDNA libraries enriched for long transcripts without size selection of insert DNAs. After cDNA synthesis, a specially designed linker (Lone-Linker “LL-Sal4”) was ligated to both ends of each cDNA. Using optimized PCR condition, we were able to amplify long tracts (average 3.0Kb, size range 1-7 Kb). We have constructed libraries from early stages of mouse embryos by this new method, and more recently, we constructed full-length enriched stem cell libraries. Results of 5’-end sequencing of these cDNA clones will be reported.


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