Plant & Animal Genome VI Conference Plant & Animal Genome VI Conference
P23
Extensive cloning of genes and markers from the porcine genome and their mapping on chromosomes have been limited, because only a few libraries are available for this purpose. We have constructed a bacterial artificial chromosome (BAC) library of porcine genomic DNA using the pBAC-Lac vector. The genomic DNA was prepared from a boar kidney primery culture cell (a cross-brad of Large White, Landrace and Duroc) and used for construction of the library.
A total of 103,488 BAC clones were isolated and oriented in 1,078 96-well microtiter plates. An average insert size of the genomic DNA in the vector was estimated to be 127 kb by examining 189 clones, implying that the library covered the porcine genome 4.5-fold. The library was arranged to screen clones with a two-step polymerase chain reaction (PCR) system. First, PCR was applied to DNA samples prepared from 22 superpools, each of which consisted of 4,704 clones or 49 plates (96 wells x 49 plates) to locate superpool(s) containing the target sequence. Then, 4-dimensional (4D) PCR is applied to DNA samples prepared from the four-dimensionally assigned BAC clones of a particular superpool.
The genome-equivalency of the library was estimated by subjecting the superpools to PCR analysis with 125 pairs of primers which represent linkage markers on 18 autosomal linkage groups, and 2 sex (X and Y) chromosomes. The number of superpools, in which a fragment specific for a particular primer pair was produced, was assumed to be a genome-equivalent of the BAC library. The number of superpools producing specific fragments per primer pair ranged from 0 to 10 with an average of 4.3. The mean value, therefore, demonstrated that the library consists of over 4.3 genome-equivalents, which is in good agreement with the calculated value based on the insert size of genomic fragments and number of clones.