ANALYSIS OF A RANDOMLY ASSORTED CDNA LIBRARY FROM BW-5147 LYMPHOID-CELLS - FREQUENCY ESTIMATES BASED ON COMPUTER-AIDED CONCATENATION MATCHING OF 2D GEL POLYPEPTIDE PRODUCTS

A cDNA library was divided into 291 sectors of low complexity, 80-1000 recombinant phage plaques per sector. Aliquots of DNA from sectors pr epared from high titer phage were subjected to in vitro transcription using T7 polymerase and thereafter RNA was translated in a cell-free r abbit reticulocyte system in the presence of [S-35] methionine. The re sulting polypeptides were separated by 2D gel electrophoresis. Radiofl uorographs of the gels prepared from 10 sectors were subjected to scan ning and image analysis. A multilevel spot matching procedure was empl oyed allowing us to recognise spot occurrence in the 10 sectors. The n umber of detected polypeptide spots per sector varied between 147 and 325. Overall, 1082 different spots were counted totalling 1983 spots c onsidering multiple entries. The distribution of the spots and the fre quency distribution of the RNA population among the 10 sectors are sho wn. The highest detected frequency is 10(-2), while one half of the RN A molecular species are present at a frequency of 10(-3) or lower. (C) 1998 Elsevier Science Ltd. All rights reserved.