Construction and Characterization of a 10-fold Genome Equivalent Rat P1-Derived Artificial Chromosome Library
- 15 June 1998
- journal article
- Published by Elsevier BV in Genomics
- Vol. 50 (3), 306-316
- https://doi.org/10.1006/geno.1998.5319
Abstract
A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614 384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.Keywords
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