|Genome Mapping Section
DOE Human Genome Program Contractor-Grantee Workshop
|64. "RPCI" Human and Mouse Bacterial Artificial
Chromosome Libraries: Construction and Characterization
Kazutoyo Osoegawa1, ChungLi
Shu1, Baohui Zhao1,
Minako Tateno2, Eirik
Frengen1, Joseph J. Catanese1,
Yoshihide Hayashizaki2 and Pieter J. de Jong1
Human male and female bacterial artificial chromosome (BAC) libraries have been constructed in the pBACe3.6 vector in compliance with the new NIH-DOE guidelines on anonymous donor selection and informed consent. A 25-fold genome equivalent male BAC library (RPCI-11) was constructed by partial digestion with a combination of EcoRI and EcoRI methylase. The library has been distributed to 30 genome centers as a major resource for the human genome sequencing effort. The average insert size was estimated to be 173 kb. The average redundancy of the library was determined at 23.9 positives per marker by hybridization of 45 single locus probes. All 1,076 marker-positive BAC clones were confirmed to be part of 45 single-marker contigs by restriction-fingerprinting. An additional 123 BAC clones were identified using probes derived from a minimal overlapping set of PAC clones on 14q24.3. The resulting 1.5-Mb BAC contig has been assembled by hybridization using BAC-end probes and PCR with STS markers, thus allowing the mapping of 264 markers within the contig. The genomic sequence for the contig region generated at Washington University, facilitates the analysis of the contig-integrity and allows the determination of the BAC clone fidelity. A total of 87 clones were confirmed to be non-chimeric because both insert-ends map back to the contig. No rearranged clones have been observed within the 5.5-kb STS-resolution contig map. More recently, a second BAC library (RPCI-13) has been constructed from an anonymous female donor. This library has been generated from genomic DNA partially digested with EcoRI (segment 1 & 2, 10x redundant) and partially digested with DpnII (segment 3 & 4, 10x redundant).
In addition to the human BAC libraries, two approximately 10-fold redundant murine BAC libraries have been prepared and extensively characterized. The source DNA for these libraries was obtained from female mice from two inbred strains: 129SvEvTAC and C57B6 for the RPCI-21 & 23 libraries, respectively. The information on the current libraries can be obtained on our Web page at: http://bacpac.med.buffalo.edu.
* Supported by grants from the U.S. DOE (#DE-FGO3-94ER61883),
NIH (#1RO1RGOl 165).
|Author Index||Sequencing Technologies||Microbial Genome Program|
|Search||Mapping||Ethical, Legal, & Social Issues|