Microdissection of human chromosomal regions 8q23.3-q24.11 and 2q33-qter: Construction of DNA libraries and isolation of their clones

Tetsuya Hirota*, Kazuhiro Tsukamoto, Han Xiang Deng, Koh ichiro Yoshiura, Tohru Ohta, Takaya Tohma, Tetsuya Kibe, Naoki Harada, Yoshihiro Jinno, Norio Niikawa

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

Human chromosomal regions 8q23.3-q24.11 and 2q33-qter were microdissected, DNAs from the regions were amplified with the primer-linker method of polymerase chain reaction (PCR), and their DNA libraries were constructed by cloning into pUC19. The primer-linker PCR involved Sau3AI digestion of microdissected chromosomal DNAs, ligation of the digests to a 10 mer DNA linker and 24 mer primer, filling the recessed 3′ ends, and PCR amplification using the 24 mer DNA as a primer. A total of 3.5 × 104 pUC19 recombinants (8q library) from the 8q region and 5.0 × 104 pUC clones (2q library) from the 2q region were obtained. From the 8q library, 60 pUC clones were selected, while 88 pUC-clones were selected from the 2q library. These clones were Southern blot analyzed on hybrid cell panels with or without human chromosome 8 or 2. Twelve (20%) of the 60 8q-derived clones were unique DNA sequences, and 9 were subjected to deletion analysis in the genomic DNA of two patients, one with trichorhino-phalangeal syndrome (TRPS) type I and the other with TRPS type II, both with del(8) (q23.3q24.13). Five of the 9 pUC clones tested showed a one-copy density in both patients, an indication that the clones map to the region deleted in both patients. Screening a genomic DNA library constructed in the phage revealed a clone with a 9.4-kb insert and a one-copy density in both patients. From the 2q library, 15 (17%) of the 88 pUC clones obtained were unique sequences. When a phage library was screened, 8 clones were obtained: 4 were identical and 2 were overlapping sequences. Using chromosome fluorescence in situ hybridization, the 4 identical sequences were mapped to 2q33.3 and the 2 overlapping clones to 2q35. These cloned DNAs will be useful in the molecular analysis of TRPS or Waardenburg syndrome type I.

Original languageEnglish (US)
Pages (from-to)349-354
Number of pages6
JournalGenomics
Volume13
Issue number2
DOIs
StatePublished - Jun 1992

Funding

We express our gratitude to Drs. Yoshimitsu Fukushima and Yoshi-fumi Yamamoto for providing blood samples of TRPS patients, Dr. Mitsuo Oshimura for providing human-mouse hybrid cell lines, A9Neo2, A9Neo8, and A9Neo15, and Dr. Tadashi Kajii for his careful review of manuscript. We also thank the American Type Culture Collection (ATCC) for giving us the total human genomic library (Catalog No. 37333). This study was supported in part by Grants-in-Aid for Scientific Research (02454493) and for Cancer Research (02262103 and 03258232) from the Ministry of Education, Science and Culture of Japan.

ASJC Scopus subject areas

  • Genetics

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