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CHORI-502: Human MHC BAC Library |
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The CHORI-502 BAC library has been
constructed by Yu Wang at BACPAC Resources, Children's Hospital
Oakland Research Institute in collaboration with other scientists
belonging to the MHC Haplotype Consortium (http://www.sanger.ac.uk/HGP/Chr6/MHC/consortium.shtml).
The human COX cell line was cultured and genomic DNA was embedded
in agarose by Dr. Simon Forbes (Department of Pathology, Cambridge
University). The agarose embedded DNA was partially digested
with a combination of EcoRI and EcoRI Methylase. Size selected
DNA was cloned into the pTARBAC2.1 vector between the EcoRI
sites. The ligation products were transformed into DH10B electro-competent
cells (BRL Life Technologies).The library has been arrayed
into 576 384-well microtiter dishes and was subsequently gridded
onto twelve 22x22cm
nylon high-density filters for hybridization screening.
Each hybridization membrane represents over 18,000 distinct
BAC clones, stamped in duplicate. Library construction was
supported through a sub-contract from The Sanger Centre.
The average insert
size of the CHORI-502 library has been determined to be 145-kb
using Pulsed Field Gel Electrophoresis. Clone
size distribution has been plotted graphically.
This 10-fold redundant
library was prepared specifically for the MHC Haplotype sequencing
project. Library copies are not available. Filters and single
clone orders can be obtained according to the distribution
policy of BACPAC Resources. The list of minimal clones spanning
the 4.5 Mbp MHC COX haplotype can be accessed as supplementary
Table 3 provided online on the Genome Research website,
accompanying the manuscript by Stewart et al. Please realize
that the clone names listed in this table, should be modified
slightly to conform with the NCBI Clone nomenclature recommendations
used for our online ordering system. An example: Clone CHORI-502-254L4
should be translated into CH502-254L4, which will be recognized
by the BACPAC electronic shopping cart. To find BAC clones
containing specific HLA loci: please consult Supplementary
Table 1. This lists the BAC clones shotgun seqenced for the
PGF halotype by their sequence accession numbers, and arranged
in linear order along the 4.5 Mbp haplotype. The conversion
from accession number to BAC clone name can be found in Supplementary
table 3. In addition to the shotgun-sequenced BAC clones,
many more BACs from the MHC region have been found but are
not listed. All MHC-region BAC clones were found by screening
high-density colony filters with synthetic overgo probes,
followed by clone fingerprinting to discover the overlap between
the clones. A minimal inventory of the high-density filters
is maintained and might be used for screening for additional
clones outside the MHC region. Screening filters and clones
can be ordered through our online ordering system.
Please visit the ordering information page if you want to
place an order.
Academic and commercial users interested in hybridization
filters of the BAC library should contact [email protected],
phone (510)450-7917, fax: (510) 450-7924), or preferably place
an order through the BACPAC online system.
Reference:
Stewart CA, Horton
R, Allcock RJ, Ashurst JL, Atrazhev AM, Coggill P, Dunham
I, Forbes S, Halls K, Howson JM, Humphray SJ, Hunt S, Mungall
AJ, Osoegawa K, Palmer S, Roberts AN, Rogers J, Sims S, Wang
Y, Wilming LG, Elliott JF, de Jong PJ, Sawcer S, Todd JA,
Trowsdale J, Beck S. Complete MHC haplotype sequencing for
common disease gene mapping. Genome Res. 14:1176-87.
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