An improved method for cloning EST-specific full-length cDNAs

Sabrina Semprini
Department of Medicine
Human Molecular Genetics Unit
University of Vermont
Health Science Research Facility 227
149, Beaumount Ave
Burlington, Vermont 05405
U.S.
telephone: 001-802-656-8921
fax: 001-802-656-8903
email: ssemprin@zoo.uvm.edu
prestype: Poster
presenter: Sabrina Semprini

S. Semprini¹, C. Collins¹, A. Colosimo², M. Pavoni³, B. Dallapiccola², G Novelli³, D.C. Gruenert¹
1.Human Molecular Genetic Unit, University of Vermont, VT USA
2.CSS Mendel Institute, Rome, Italy
3.Dept. of Biopathology and Diagnostic Imaging, Tor Vergata University of Rome, Rome Italy

Primary approaches for cloning full-length cDNAs involve cDNA library screening and rapid amplification of cDNA ends (RACE). These techniques are laborious and time consuming. Recently, a technique developed to clone expression sequence tag (EST)-specific full-length cDNA has streamlined this process considerably. This strategy is based on EST-specific mRNA enrichment with EST-derived biotin labeled "capture" primers and streptavidin-coated magnetic beads. Enriched mRNA is then used to generate cDNA for cloning into plasmid vectors. This strategy has now been further modified to improve its efficiency and specificiy for cloning full-length cDNA. This modification involves the ligation of a double-stranded DNA (dsDNA) adapter to a RNA/cDNA hybrid molecule. As a proof-of-principle, first strand cDNA synthesis was performed on kanamycin mRNA using a Not I oligo(dT)15 primer. The second adapter, containing an MluI restriction site, was ligated to the mRNA-cDNA hybrid. Following adapter addition, a second strand of cDNA was synthesized. Resultant (ds)cDNAs were size fractionated prior to ligation into the pSPORT I vector. Bacterial cells were transformed with the vector with the cDNA insert. Resultant colonies were screened by PCR. Sequence analysis showed full-length kanamycin sequence in positive clones. These results indicate the potential of this technique in the rapid cloning of full-length cDNAs. This improved technique has now been applied to isolating full-length sequences corresponding to breast cancer candidate ESTs, using a novel cDNA cloning vector that optimizes the cloning of large cDNA inserts.

This work was founded by NIH Grant #CA80200-03