RPCI - 11 Human Male BAC Library
The RPCI-11 Human Male BAC Library (Osoegawa et al., 2001) was constructed using improved cloning techniques (Osoegawa et al., 1998) developed by Kazutoyo Osoegawa. The library was generated by Kazutoyo Osoegawa. Construction was funded by a grant from the National Human Genome Research Institute (NHGRI, NIH) (#1R01RG01165-03). This library was generated according to the new NHGRI/DOE "Guidance on Human Subjects in Large-Scale DNA Sequencing".
Male blood was obtained via a double-bind selection protocol. Male blood DNA was isolated from one randomly chosen donor (out of 10 male donors) and partially digested with a combination of EcoRI and EcoRI Methylase. Size selected DNA was cloned into the pBACe3.6 vector between the EcoRI sites. For Segment 5, the same male donor DNA was partially digested with MboI, size selected, and ligated into the pTARBAC1 cloning vector at the BamHI sites. The ligation products were transformed into DH10B electrocompetent cells (BRL Life Technologies). The library has been arrayed into 384-well microtiter dishes and also gridded onto 22x22cm nylon high density filters for screening by probe hybridization.
The RPCI Human Male BAC Library:
Segment |
DNA |
Plate Numbers |
Total Plates |
Total Clones |
Empty Wells (Total) |
|
1 |
pBACe3.6 (1) |
Male | 1-288 | 288 | 108,499 | 2,093 |
2 |
pBACe3.6 (1) | Male | 289-516 | 288 | 109,496 | 1,096 |
3 |
pBACe3.6 (1) | Male | 577-864 | 288 | 109,657 | 935 |
4 |
pBACe3.6 (1) | Male | 865-1152 | 288 | 109,382 | 1,210 |
5 |
pTARBAC1 (2) | Male | 1153-1440 | 288 | 106,763 | 3,289 |
Total Library |
1-1440 | 1440 | 543,797 | 9,163 |
1: donor DNA EcoRI partially digested
2: donor DNA MboI partially digested
| Segment | Empty Wells (%) | Non-Recombinant Clones (Total) | Non-Recombinant Clones (%) | Insert Size (average) | Genomic Coverage |
| 1 | 1.9 | approx. 1800 | 1.7 | 164 Kbp | 5.8X |
| 2 | 1.0 | approx. 550 | 0.5 | 168 Kbp | 6.0X |
| 3 | 0.8 | approx. 1100 | 1.0 | 181Kbp | 6.7X |
| 4 | 1.1 | approx. 1100 | 1.0 | 183Kbp | 6.8X |
| 5 | 3.5 | approx. 530 | 0.5 | 196Kbp | 6.9X |
| Total Library | 1.7 | approx. 5080 | 0.9 | 178 Kbp | 32.2X |
click here for a legend of the previous tables.
The average insert size has been
determined by Pulsed Field Gel Electrophoresis analysis of clones randomly chosen
from plates from each segment.
Reference:
1. Osoegawa, K., Mammoser, A. G., Wu, C., Frengen, E., Zeng, C., Catanese, J. J., de Jong, P. J. (2001) A Bacterial Artificial Chromosome Library for Sequencing the Complete Human Genome, Genome Research, Vol. 11, Issue 3, 483-496, March 2001 (Reprints available upon request)
2. Osoegawa, K., Woon, P.Y., Zhao, B., Frengen, E., Tateno, M., Catanese, J.J, and de Jong, P.J. (1998) An Improved Approach for Construction of Bacterial Artificial Chromosome Libraries, Genomics 52, 1-8; Article # GE985423. (Reprints available upon request)