Genomic libraries

Genomic libraries used and the details are described below.

(i) Human IC I Yeast Artificial chromosome (YAC) library

The human ICI YAC library (Anand et al., 1990) was supplied by the HGMP resource centre (Cambridge, UK). The library was made from a human lymphoblastoid 48XXXX cell line, and contains approximately 34,500 recombinant clones. The average insert size is 350Kb and so far data suggest that approximately 10% o f the clones are chimaeric. The ICI YAC library is composed o f three sets o f YAC pools, the primary, the secondary and the tertiary. The primary ICI YAC pool consists of 40 pools o f YAC clones, and each primary set o f ICI YAC pools is split into 9 aliquots, labelled A to I, which constitute the secondary YAC pool. An individual pool from the secondary set is categorised into an additional 20 pools, the tertiary pool. The first eight sets are labelled A to H, and the rest 9 to 20. These 20 sets o f YAC clones, in the form o f plugs, form a grid system. The YAC library was screened through the primary to the tertiary pools o f decreasing complexity until a single positive clone was identified. Refer to Anand et al. (1990) for interpretation o f positive results.

The individual YAC DNA clones o f the primary, secondary and tertiary YAC pools are

Methods and Materials stored in agarose plugs in Ix TE buffer (pH8.3) and 5mM EDTA (pH8.3). One half o f each YAC plug representing each o f the pools was cut in half and washed w ith SDW for 2 hours, to remove all inhibitory reagents from the agarose. The plugs were then placed in a new 1.5ml Eppendorf tube and melted at 65°C for 5 minutes. An equal volume o f SDW was added to each o f the tubes. 2-3p.l o f this aliquot o f YAC clone DNA was used for the PCR amplification, using gene specific primers.

('ii) Human genomic cosmid library

A n aliquot o f the human genomic cosmid library was acquired from University College London (UCL) (Cachon - Gonzalez, PhD thesis 1991). The library had been constructed in the Lorist B cosmid vector and consists o f approximately 5 x 1 0 ^ colony forming units (cfu) per til. The cloning vector carries the kanamycin resistance gene.

Ciii) Pig genomic cosmid library

The pig genomic cosmid library was kindly donated by Dr. M. Varman, Laboratoire de Radiobiolgie A ppliquée, 78352, Jouy-en-Josas, France. This library had been constructed from genomic DNA taken from a genotypically SLA homozygous large white individual. The genomic DNA had been isolated from a fresh pig spleen, partially digested with the restriction enzyme Sau 3A and cloned into the Bam HI site o f the Lorist B cosmid vector. The vector carries the antibiotic resistance gene kanamycin. The titre o f the library was estimated to be approximately 2.9 x 10^ cfu per ml. E. Cali strain ED8767 had been used for amplification.

('iv) Chicken genomic cosmid library

This library was purchased from Clontech Laboratories (USA). The library had been constructed using genomic DNA extracted from adult Leghorn male liver, digested w ith Bam H I and cloned into pWE15 cosmid vector. The library had been amplfied in E. coli NM554 cells and the titre o f the library had been estimated to be greater than 10^ cfu/ml. The insert size was reported to ranged between 35-50Kb, with an average size o f 38Kb. The cosmid vector confers resistance to ampicillin.

(v) 129 S V J Mouse genomic phage library

This phage library, from Stratagene, USA, had been constructed using DNA extracted

Methods and Materials from the liver o f a 4-8 weeks old female. The genomic DNA had been partially digested with Sau?> A l and cloned into the Xho I site o f the Lambda FIX® II vector. The insert size was reported to range between 9-23Kb. The titre was estimated to be 2.0 x 10^® plaque forming units (pfu) per ml and the amplification host strain is XL 1-Blue MRA(P2) cells. This strain o f E.co// was also obtained from Stratagene.

(vi) Human genomic P I Artificial Chromosome (PAC) library

PACs are bacteriophage PI-derived artificial chromosome based vectors which use the bacteriophage T4 in vitro packaging system (loannou et a l, 1994). Recombinant PAC clones do not suffer from chimaerrism and cloned inserts have been reported to be stable after extended culturing. The human genomic PAC library-RPCIl was constmcted by Pieter de Jong'g group at the Poswell Park Cancer Institute, Buffalo (unpublished). The library had been constmcted using DNA from a normal male donor, cloned into vector pCYPAC2N. The library consists o f approximately 1.2 x 10^ clones and it has been reported that approximately 25% o f these may lack human DNA inserts. The library itself is maintained in 321 microtitre plates (384-well format), grown in LB medium supplemented with kanamycin (25|Lig/ml) and 7% glycerol. The library had been gridded in 4x4 array onto 22cm^ Hybond N+ nylon membranes. Each clone has been spotted twice, giving a total o f 36,864 clones (18,432 x 2) on each membrane. Seven high density gridded membranes represent the whole library. These were supplied by the Human Genome Mapping Project (HGMP) resource centre, UK. Instmctions for the usage o f the library membranes and the interpretation o f the hybridisation results were also supplied by the resource centre.

(vii) Mouse genomic P I Library

This library had been constmcted using genomic DNA derived from C57BL/6 female mice. The entire library is represented on 7 Hybond N+ membranes. The genomic DNA fragments had been ligated into pAdlOSacBII vector (Pierce, J. C et a l, 1992) and the recombinant DNA had been transformed into the bacterial host strain NS3145 (unpublished). The vector carries the kanamycin resistance gene. The membranes were obtained from the Genome Analysis Laboratory, Imperial Cancer Research Fund (ICRF, UK). Each membrane had been organised into 48 by 48 boxes, each box containing 16 clones (8 clones in duplicate) making a total o f 36, 864 duplicate clones. Instmctions for the usage o f the library membranes and the interpretation o f the hybridisation results

Methods and Materials were supplied with the membranes.