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CHORI-507: Human chromosome 21 specific library (WAV17 (HC21p project)) BAC Library |
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Summary.
We prepared a BAC library from the somatic cell hybrid WAV17 Slate et al. 1978, which contains only human chromosome 21 (HSA21), derived from a single HSA21, in a mouse background. This ensures that derived non-mouse BACs map to human chromosome 21. To ensure the integrity of WAV17, we karyotyped the line and used FISH to confirm the human chromosomal content (See Fig. 1, Supplement to publication). WAV17 was also genotyped for six HSA21 microsatellite markers and only one allele was observed in each case, confirming the monoallelic composition of HSA21 in WAV17. The purpose of the library construction was to identify BAC clones from the short arm of human chromosome 21. In the intitial phase 576 microtiter dishes (384-well) type were obtained with ~ 220,000 individual clones. Following screening with human=specific repeat probes, 3,208 putative human BACs were identified and re-arrayed into secondary microtiter dishes. Candidate 21p clones were identified by direct screening with specific probes and also by elimination of chromosome 21q BACs following BAC-end-sequencing and alignment with chromosome 21q. The results have been published Lyle et al, Genome Res. 2007 17:1690-6
BAC Library Preparation for Hybrid Cells.
The CHORI-507 BAC library was prepared as described Osoegawa et al. 1998. Briefly,
high-molecular-weight DNA from WAV 17 cells was prepared in agarose using DNA Plug Kits (BioRad). The DNA was partially digested with MboI (New England Biolabs) to generate fragments between 100 kb and 400 kb, which were size-fractionated by pulsed-field electrophoresis [Osoegawa et al. 1998]. DNA fractions in the 150 to 250 kb range were sliced from the gel and DNA was electro-eluted from the gel matrix. Vector DNA pTARBAC1.3 was prepared by treatment with a combination of restriction enzymes (ApaLI and BamHI) and alkaline phosphatase and then purified by gel-electrophoresis. E. coli DH10B T1 phage-resistant cells (Invitrogen) were transformed by electroporation with ligation products from vector and genomic DNA. Prior to full-scale library production, 379 random clones were picked for DNA size analysis. BAC DNA, prepared using an Autogen-960 robot, was digested with NotI (New England Biolabs), and analyzed by pulsed-field electrophoresis in a 1% agarose gel. The library clone size distribution is given in Supplemental Fig. 2, and the average insert size was estimated to be 141 kb. There is a small fraction (1 out of 379) of non-recombinant clones. Following this quality assessment, 221,184 BAC clones (approximately 10-fold coverage) were arrayed into 576 384-well microtiter plates. Duplicate sets of twelve high-density colony filters were created for hybridization screening of the library with various human-specific repeat probes.
Screening BAC library CHORI-507 for human BAC clones.
To identify human clones, CHORI-507 was screened with 32P labeled "-satellite and Alu probes, and total human genomic DNA. This resulted in 2400 (1.09%) positive clones after screening using "-satellite and Alu probes, and 2752 (1.24%) positives with total human genomic DNA. This is consistent with the fact that the HSA21 content of WAV 17 is approximately 1-3% in an aneuploid mouse background. The non-redundant positive clones (3,208) were re-arrayed into 384-well plates and gridded on nylon membrane for screening with 21p-specific probes. To identify specific 21p BACs, overgo probes were designed to STSs corresponding to the YAC ends 2E4R, 2E4L, 4E9L, 4E9R, 1B8L, 2C2R, 2C9R from Wang et al. [Wang et al. 1999]. A summary of the library screens is given in Supplemental Table 1 and Supplemental Table 2.
BAC end sequencing
Positive clones from CHORI-507 were end sequenced using the following protocol. BAC clones were inoculated into 1.5ml of 2xTY containing 12.5 μg/ml chloramphenicol in 2ml 96 well growth boxes (Costar) and incubated for 22 hours at 37°C with shaking at 320 rpm. Plates were centrifuged at 4000rpm for 3 min to obtain pellets, the supernatant was discarded and the cells were re-suspended in 100μl of GTE+RNaseA, 100μl of NaOH/SDS was added and mixed before adding 100μl of 3M KOAc and further mixing. After filtration through two Millipore plates (MADVN6550/MANUBA50) on a vacuum manifold, the resultant material was dried and re-suspended in 35μl of 10mM Tris pH8.0. The resulting DNA was sequenced with BigDye Terminator Ready Mix v3.0 and BigDye Bufferx5 (Applied Biosystems) using T7 and SP6 primers. The sequenced products were cleaned up by washing with 5μl 3M NaOAc and 125μl 96% ethanol. The DNA was precipitated by centrifugation at 4000rpm for 15 mins, then washed with a further 100μl 70% ethanol and the centrifugation repeated. The products were re-suspended in 10μl of 0.1M EDTA, pH 7.4 and loaded on to ABI3700 capillary sequencers.
Fingerprinting of BAC clones
BAC clones were fingerprinted using HindIII restriction enzyme fingerprinting [Humphray et al. 2001; Schein et al. 2004]. BAC clones were directly cultured in 170μl 2xTY in 96 well plates. After overnight growth the BAC DNA was extracted by alkaline lysis, and digested with HindIII. Following electrophoresis on 121 lane 1% agarose gels the data are collected using a Typhoon 8600 fluorimager, raw images were entered into the fingerprint database using IMAGE oftware(http://www.sanger.ac.uk/Software/Image). The output of normalized band values, sizes and gel traces were analyzed in FPC [Soderlund et al. 2000], which bins and orders clones on the basis of shared bands. Sequence tilepaths were identified following inspection of assembled contigs. End sequences were aligned to the human and mouse
genomes using BLAST and ssaha2 (www.ensembl.org).
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