| Project/Area Number |
02454493
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Human genetics
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| Research Institution | Nagasaki University School of Medicine |
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Principal Investigator |
NIIKAWA Norio Nagasaki University School of Medicine, Department of Human Genetics, Professor, 医学部, 教授 (00111170)
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| Co-Investigator(Kenkyū-buntansha) |
JINNO Yoshihiro Nagasaki University School of Medicine, Department of Human Genetics, Instructor, 医学部, 助手 (20179097)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1991: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1990: ¥3,700,000 (Direct Cost: ¥3,700,000)
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| Keywords | Chromosome / Microdissection / DNA Loning / DNA / Library / Chromosome Painting / Gene Mapping / Human Genome / 染色体ミクロ切断 / 未知遺伝子 / 染色体バンド特異的DNAクロ-ン / in situ hybridization / PCR / 遺伝子クロ-ニング |
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| Research Abstract |
In order to construct chromosomal region or band-specific DNA libraries, we developed a method of chromosome microdissection/microcloning. In short, a defined chromosomal region or band was dissected under a microscope equipped with a fine glass-needle handling a micromanipulator. From several chromosome pieces dissected and collected, DNA was extracted and ligated to a 10mer DNA (linker)/24mer DNA (primer) under a microscope. PCR was performed by 30 cycles, using the 24mer DNA as a primer. PCR amplified DNA was ligated to pUC19 and cloned.
A total of 35, 000 clones were obtained from a region 8q23.3-q24.11, and 50, 000 from a region 2q33-qter. The confirmation that the PCR product was derived from the dissected regions was done by fluorescence chromosome in situ suppression hybridization (chromosome painting) using the product as a probe pool. Of 60 clones selected from the 8q-library, 12 (20%) were unique (single-copy) sequences, while 15 (17%) of 88 clones from the 2q-library were unique sequences. Nine of the 12 unique clones from the 8q-library showed a one-copy density in two patients with tricho-rhino-phalangeal syndrome (TRPS) and del (8) (q23.3q24.12), an indication that they are mapped to the region deleted in both patients. Screening of a phage-libraty with the 2q-specific microclones revealed 6 clones and they are mapped at the dissected region. Of the 6 clones, 2 revealed RFLPs. These clones are useful for analaysis of TRPS or Waardenburg syndrome type 1.
The microdissection/PCR was applied to the diagnosis of chromosome abnormalities of which origin was unknown. With this technique, an minute additional marker chromosome was successfully traced ; it was derived from Y chromosome. Likewise, an additional segment of 17p+ was traced to be derived from 15qter.
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