Showing papers by "Ileana Zucchi published in 1994"

Rapid isolation of cDNA clones by aliquot testing via PCR amplification.

Abstract: Istituto di Tecnologie Biomediche Avanzate, Consiglio Nazionale delle Ricerche, Milan, Italy In the last few years our laboratory has been involved in the identification and sequencing of genes in Xq24-qter. (1-4) During this work, a large number of data was obtained that could be analyzed with ad hoc software tools. From this analysis it was possible to identify specific fragments that had a high probability of containing coding sequences; however, direct proof of the existence of transcript still needed the labor-intensive approach of screening the cDNA libraries. In addition, the pattern of tissue expression of these putative transcripts was not known beforehand, and several tissues had to be tested, via Northern analysis, to detect the appropriate cell type, developmental stage, or tissue of expression. Here, we report a rapid PCR-based method of identifying cDNAs from genomic sequences with a high probability of being transcribed. Prescreening cDNA libraries by PCR amplification with primers designed in putative exons is an efficient way of determining which library has to be screened for the isolation of the desired mRNAs. Additionally, performing PCR assays on aliquots of a discrete number of cDNA clones from nonamplified libraries, instead of performing traditional hybridization screening directly on millions of phages, is both fast and economical and allows the systematic screening of cDNA libraries too, as it has been reported for YAC and k genomic DNA libraries. (s'6) These aliquots can be stored easily, used for an indefinite number of screenings, and maintained for an indeterminate period of time. The setting up of this procedure and its application using three cDNA libraries will be discussed.