Human bac library: its application to mapping of 21q22.3 and isolation of disease genes

We have constructed a human genomic BAC library using a human pre-pro-B cell line. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. We have established three different screening systems: 1) Probe hybridization to 31 high density replica (HDR) filters: each filter contains 3,072 BAC clones which were gridded in a 6 x 6 pattern, 2) Probe hybridization to 2 Southern blot filters each containing 31 Hind III digests of the pooled 3,072 BAC clones. This identifies a particular HDR filter for further probe hybridization, and 3) Two step-polymerase chain reaction (PCR): First PCR is applied to 10 DNA samples from superpools of 9,600 BAC clones to identify a particular superpool and the second PCR applies to 40 DNA samples from 4 dimensionally assigned BAC clones. The two step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The BAC clones are very useful to construct long range contigs and to utilize starting as a material of sequencing analysis. As a typical example of BAC application, the analysis of 21q22.3 has been presented.