Marcelo Bento Soares, Kala Mayur, Maria de Fatima Bonaldo and Susan Baumes
Department of Psychiatry, Columbia University and The New York State Psychiatric Institute, New York, NY 10032
We have further optimized our original procedure for construction of normalized directionally cloned cDNA libraries and we have successfully applied it to generate human cDNA libraries from fetal liver-spleen, full-term and 8-9 week placentae, adult breast, brain, retina, pineal gland, ovary tumor, melanocytes and multiple sclerosis plaques. Several additional libraries are currently in preparation. All libraries have been contributed to the IMAGE consortium, and they are being widely used for sequencing and mapping.
Several milestones have been accomplished towards our final objective of generating full-length normalized cDNA libraries. First, we have adapted our normalization protocol to take advantage of the fact that it is now possible to produce single-stranded circles in vitro by sequentially digesting supercoiled plasmids with Gene II protein and Exonuclease III (Life Technologies). This is significant because it circumvents the biases introduced by differential growth of clones containing small and large cDNA inserts when single-strands are produced in vivo upon superinfection with a helper phage. Second, a number of parameters have been optimized regarding synthesis of first and second strand cDNA. Third, we have optimized conditions for size selection of RNA and we have generated several size fractions of a pool of placentae and brain mRNAs. We are currently starting to construct cDNA libraries from individual RNA size fractions. The fact that the size range of the starting template RNA population is known makes it possible that we perform a strict size selection of the synthesized double-stranded cDNAs and thereby greatly enrich for full-length molecules prior to cloning.
Supported by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-FG02-91 ER61233
 Soares, M.B. , Bonaldo, M.F., Su, L., Lawton, L. & Efstratiadis, A. Construction and characterization of a normalized cDNA library (1994). Proc. Natl. Acad. Sci. USA 91(20), 9228-9232.
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