Human bac library: its application to mapping of 21q22.3 and isolation of disease genes

Shuichi Asakawa, Izumi Abe, Yoshiki Kudoh, Kentaro Nagamine, Yimin Wang, Kazuhiko Kawasaki, Jun Kudoh, Shinsei Minoshima, Nobuvoshi Shimizu

Research output: Contribution to journalArticle

Abstract

We have constructed a human genomic BAC library using a human pre-pro-B cell line. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. We have established three different screening systems: 1) Probe hybridization to 31 high density replica (HDR) filters: each filter contains 3,072 BAC clones which were gridded in a 6 x 6 pattern, 2) Probe hybridization to 2 Southern blot filters each containing 31 Hind III digests of the pooled 3,072 BAC clones. This identifies a particular HDR filter for further probe hybridization, and 3) Two step-polymerase chain reaction (PCR): First PCR is applied to 10 DNA samples from superpools of 9,600 BAC clones to identify a particular superpool and the second PCR applies to 40 DNA samples from 4 dimensionally assigned BAC clones. The two step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The BAC clones are very useful to construct long range contigs and to utilize starting as a material of sequencing analysis. As a typical example of BAC application, the analysis of 21q22.3 has been presented.

Original languageEnglish
Number of pages1
JournalJapanese Journal of Human Genetics
Volume42
Issue number1
Publication statusPublished - 1997 Dec 1

ASJC Scopus subject areas

  • Genetics(clinical)

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    Asakawa, S., Abe, I., Kudoh, Y., Nagamine, K., Wang, Y., Kawasaki, K., Kudoh, J., Minoshima, S., & Shimizu, N. (1997). Human bac library: its application to mapping of 21q22.3 and isolation of disease genes. Japanese Journal of Human Genetics, 42(1).