A 3.5 genome equivalent multi access YAC library: construction, characterisation, screening and storage

Abstract

The construction of a yeast artificial chromosome (YAC) primary gridded library of 35,000 clones from human lymphoblastoid (48,XXXX) cell line DNA is described. The average YAC size is approximately 350kb representing a greater than 3.5 times coverage of the genome. The library is stored at -70 degrees C as gridded clones on nylon filters impregnated with 20% glycerol and as glycerol suspensions of individual clones in microtitre plates providing a prolonged multi-user potential. To date we have used 14 single copy probes to screen this library by colony hybridisation as well as PCR and have isolated between 1 and 5 YAC clones for every probe.

Chromosomal localization of human genes for arylamine N-acetyltransferase

“…Screening of the YAC library (Anand et al, 1990) was carried out on pools, as supplied by the Human Genome Mapping Project (HGMP) Resource Centre, using PCR with primers specific for AAC-J and AAC-2. Specificity of amplification was confirmed by restriction enzyme digestion, with HincII, or its isoschizomer Hindll, and HindlIl digesting AAC-2 and AAC-J respectively .…”

A novel human Wnt gene, WNT10B, maps to 12q13 and is expressed in human breast carcinomas

“…The WNT10B gene was found to be located on chromsome 12 (data not shown). The same PCR approach was used to screen the ICI YAC library (UK HGMP Resource Centre) using conventional methods (Anand et al, 1990). Two positive YAC clones were identi®ed (26BB4, 28AD4) to contain the WNT10B gene and used in the¯uorescence in situ hybridisation (FISH) to metaphase spreads as described (Fantes et al, 1992).…”

Isolation of a Yeast Artificial Chromosome Contig Spanning the Greig Cephalopolysyndactyly Syndrome (GCPS) Gene Region

“…YAC libraries were obtained as DNA pools from the Centre d'Etude du Polymorphisme Humain (CEPH; Albertsen et al, 1990), from the Imperial Cancer Research Fund (ICRF; Larin et al, 1991), and from the Human Genome Mapping Project Resource Center, Harrow (ICI library; Anand et al, 1990). These libraries were screened with primer pairs 5-23-19 (5'-CCTGGGAAG-GTATTACTGC) and 5-23-20 (5'-GTGACTATGAATCATCAGCG) for probe p5-23 and GLldistA (5'-TCTAATCTGCAGAATTACTC) and GLIdistE (5'-TGAAACAGATCTCAGTTAGG) for the 3' part of the GLI3 gene (Vortkamp et al, 1991b; these primers amplify 580 bp of the GLI3 cDNA and also the corresponding genomic DNA).…”

“…In a PCR-based approach we screened pools of the CEPH, ICRF, and ICI YAC libraries (Anand et al, 1990;Albertsen et al, 1990;Larin et al, 1991) using primers from the distal part of GLI3 and the flanking probe p5-23. The latter probe defines the CpG island on the telomeric side of a 550-kb NotI fragment that is detected by the GLI3 cDNA.…”