Evaluation of vector-primed cDNA library production from microgram quantities of total RNA.
cDNA sequences are important for defining the coding region of genes, and full-length cDNA clones have proven to be useful for investigation of the function of gene products. We produced cDNA libraries containing 3.5-5 x 10(5) primary transformants, starting with 5 mug of total RNA prepared from mouse pituitary, adrenal, thymus, and pineal tissue, using a vector-primed cDNA synthesis method. Of approximately 1000 clones sequenced, approximately 20% contained the full open reading frames (ORFs) of known transcripts, based on the presence of the initiating methionine residue codon. The libraries were complex, with 94, 91, 83 and 55% of the clones from the thymus, adrenal, pineal and pituitary libraries, respectively, represented only once. Twenty-five full-length clones, not yet represented in the Mammalian Gene Collection, were identified. Thus, we have produced useful cDNA libraries for the isolation of full-length cDNA clones that are not yet available in the public domain, and demonstrated the utility of a simple method for making high-quality libraries from small amounts of starting material.
Kuo
Jonathan
J
Laboratory of Genetics, National Institute of Mental Health Building 35, Room 1B-215, 35 Convent Drive, Bethesda, MD 20892-3728, USA.
Inman
Jason
J
Brownstein
Michael
M
Usdin
Ted B
TB
D000818:Animals; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D012313:RNA / Q000378:metabolism; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D012313:RNA