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RPCI-3: Human (M) PAC Library |
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INTRODUCTION:
The "RPCI" Human PAC libraries have been constructed in our laboratory during 1993-1995 to support the initial phase of the Human Genome Project (HGP). Sequences derived from the male PAC libraries represent about 9.3% of the sequence in the reference human genome assembly. Two anonymous DNA blood samples have been used for the human PAC libraries: a male sample for libraries RPCI-1, -3, -4, AND -5 and a female sample for the RPCI-6 PAC library, respectively. The anonymous samples were obtained by a protocol which was approved by the RPCI Institutional Review Board (IRB). However, the donor selection was judged insufficiently anonymous by the NIH, as described in an editorial in Science in 1996. Following this judgement, the human "RPCI" human PAC libraries were phased out as intermediate resources for the Human GenomeProject after a one-year grace period, which ended in the Summer of 1997. This grace period allowed for the preparation of additional libraries with donors obtained through a double-blind selection strategy (see the RPCI-11 BAC library page). Nevertheless, 9.3% of the draft human genome assembly, table I was derived from the male PAC libraries: 2.8%, 1.6%, 2.2% and 2.7% from RPCI-1, -3, -4 and -5, respectively.
STRATEGY:
All PAC libraries were prepared using the same cloning approach: high-molecular weight genomic DNA was stabilized by embedding in agarose and was then partially digested with MboI. Size-fractionated genomic fragments, obtained by pulsed-field electrophoresis (CHEF system), were cloned between the BamH1 sites of either the pCYPAC2 (RPCI-1, 2, 3, 4, and 5) or the pPAC4 (RPCI-6) vector. The ligation products were transformed into DH10B electrocompetent cells (BRL/Life technologies). The PAC colonies ("clones") have been picked into a large numbers of 384-well microtiter dishes using a colony picking robot. The procedure for preparing the PAC libraries has been described by Ioannou et al 1994. The "Ioannou" publication refers to a set of PAC clones used in a pilot project to develop the cloning strategy. However, the clones described in the publication are not part of any distributed library.
CHARACTERIZATION:
The Library segments have been characterized as to:
- The percentage of empty wells
- Percentage of non-recombinant clones
- Average insert size
- Approx. genomic coverage
The clone size distributionfor each segment of the library has also been determined.
AVAILABILITY:
The library is available for individual clone orders, distributed as agar-stab cultures at a not-for-profit cost. See our webpage "Ordering & Pricing Information". Academic and commercial users interested in a copy of the PAC library should contact Pieter J. de Jong ([email protected], fax: (510) 450-7924) (subject to availability and distribution policy. For questions about clone orders, please contact BACPAC Resources ([email protected]). ACKNOWLEDGEMENTS:
The "RPCI" Human PAC libraries have been constructed by Panos A. Ioannou* (RPCI-1), Chira Chen and Baohui Zhao (RPCI-3), and Baohui Zhao (RPCI-4, 5, and 6). Library construction was supported by grant funding to our laboratory by the U.S. Department of Energy (#DE-FG02-94ER61883) and the National Human Genome Research Institute (NHGRI, NIH) (#1R01RG01165).
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