| Project/Area Number |
62480251
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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 |
内分泌・代謝学
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| Research Institution | Kyoto Univercity |
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Principal Investigator |
YOSHIKATSU Nakai Kyoto University, 医学部, 講師 (10115892)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1988: ¥500,000 (Direct Cost: ¥500,000)
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| Keywords | Apolipoprotein E / Point mutation / Hyperlipidemia / DNA amplification / Polymerase Chain Reaction / synthetic oligonucleotide / RNA probe / 遺伝子型 / 相補性DNA / 高脂血症 / ライポプローブ / 制限酵素 / SP64プラスミッド / βーVLDL |
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| Research Abstract |
A rapid procedure for determing apolipoprotein E genotype grom genomic DNA has been deceloped.
1) Single base substitutions can be detected and localized by a simple and rapid method that involves ribonuclease cleavage of single base mismatches in RNA:DNA heteroduplexes. The feasibility of this method for localizing base substitutions directly in genomic DNA samples is demonstrated by the detection of single base mutations in DNA obtained from individuals with type III hyperlipidemia, a genetic disorder in apolipoprotein E gene expression.
2) DNA is amplified by the method for in vitro DNA amplification (Polymerase Chain Reaction). Two sets of oligonucleotides were synthesized and used to discriminate either between epsilon3 and epsilon4 alleles of between epsilon3 and epsilon2 alleles. Combination of the allele-specific oligonucleotide hybridization with the method for in vitro DNA amplification dramatically improved the sensitivity and the releability of the procedure.
Adaptation of a simple strategy involving direct coloning and DNA sequencing of in vitro amplified DNA enables rapid identification of any mutation within the apo E gene area encoding the receptor binding domain.
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