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Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labeled via linear amplification.

作者信息

Cole C G, Patel K, Shipley J, Sheer D, Bobrow M, Bentley D R, Dunham I

机构信息

Paediatric Research Unit, United Medical School, Guy's Hospital, London, United Kingdom.

出版信息

Genomics. 1992 Dec;14(4):931-8. doi: 10.1016/s0888-7543(05)80114-4.

Abstract

The ability to identify large numbers of yeast artificial chromosomes (YACs) specific to any given genomic region rapidly and efficiently enhances both the construction of clone maps and the isolation of region-specific landmarks (e.g., polymorphic markers). We describe a method of preparing region-specific single-stranded hybridization probes from Alu element-mediated polymerase chain reaction (Alu-PCR) products of somatic cell hybrids for YAC library screening. Pools of up to 50 cloned Alu-PCR products from an irradiation-reduced hybrid containing 22q11.2-q13.1 were labeled to high specific activity by linear amplification using a single vector primer. The resulting single-stranded probes were extensively competed to remove repetitive sequences, while retaining the full complexity of the probe. Extensive coverage of the region by YACs using multiple probe pools was demonstrated as many YACs were detected more than once. In situ analysis using chosen YACs confirmed that the clones were specific for the region. Thus, this pooled probe approach constitutes a rapid method to identify large numbers of YACs relevant to a large chromosomal region.

摘要

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