Inexpensive, rapid and convenient PCR-minigel SSCP protocol for polymorphisms and mutations analyses of LDL receptor gene.

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dc.contributor.authorPongrapeeporn, K Sen_US
dc.contributor.authorThepsuriyanon, Pen_US
dc.contributor.authorLeowattana, Wen_US
dc.contributor.authorOng-Ajyooth, Sen_US
dc.contributor.authorKiartivich, Sen_US
dc.contributor.authorYamwong, Pen_US
dc.contributor.authorKasemsuk, Len_US
dc.contributor.authorKerdsaeng, Ken_US
dc.contributor.authorNuchpramool, Wen_US
dc.contributor.authorLaungsuwan, Sen_US
dc.contributor.authorAmornrattana, Aen_US
dc.date.accessioned2009-05-27T20:58:33Z
dc.date.available2009-05-27T20:58:33Z
dc.date.issued2001-12-11en_US
dc.descriptionChotmaihet Thangphaet.en_US
dc.description.abstractHypercholesterolemia has been recognized as a major risk factor of atherosclerosis and coronary artery disease. The elevation in plasma low density lipoprotein (LDL) cholesterol is frequently due to genetic alteration at the genetic locus specifying the LDL receptors, leading to defective catabolism of LDL. In order to facilitate the molecular diagnosis of LDL receptor disorder, single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) amplified genomic DNA fragments has become a simple and sensitive screening method for identification of DNA polymorphisms and mutations in LDL receptor gene prior to DNA sequencing. In addition, SSCP patterns can be detected by silver staining to avoid hazardous radioactive material or other costly nonradioactive detection techniques. However, the original SSCP protocol is generally large-formatted, which is both time and reagents consuming as well as cumbersome. Minigel SSCP protocols have thus been devised but they involve, although commercially available, costly precast gels. We describe here a nonradioactive PCR-minigel SSCP protocol which is sensitive, inexpensive, rapid, reproducible and manually convenient. The results in this study demonstrate that minigel-SSCP (gel size: 10 cm x 7.3 cm x 0.075 cm) can detect conformation polymorphisms in PCR-fragments with a comparative sensitivity to large gel SSCP (gel size: 30 cm x 40 cm x 0.04 cm) as exemplified by the SSCP analyses of exon 13 of the LDL receptor gene. For minigel SSCP, the reagents for gel components and silver staining are reduced approximately 9 times and 10 times, respectively. For electrophoresis, electrical power is also reduced 10 times. This improved technique can become routinely used for molecular diagnosis of LDL receptor defect as well as for other genetic disorders.en_US
dc.description.affiliationDepartment of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.en_US
dc.identifier.citationPongrapeeporn KS, Thepsuriyanon P, Leowattana W, Ong-Ajyooth S, Kiartivich S, Yamwong P, Kasemsuk L, Kerdsaeng K, Nuchpramool W, Laungsuwan S, Amornrattana A. Inexpensive, rapid and convenient PCR-minigel SSCP protocol for polymorphisms and mutations analyses of LDL receptor gene. Journal of the Medical Association of Thailand. 2001 Dec; 84 Suppl 3(): S676-83en_US
dc.identifier.urihttps://imsear.searo.who.int/handle/123456789/43971
dc.language.isoengen_US
dc.source.urihttps://www.mat.or.th/journal/all.phpen_US
dc.subject.meshDNA Mutational Analysisen_US
dc.subject.meshHumansen_US
dc.subject.meshHypercholesterolemia --geneticsen_US
dc.subject.meshPolymerase Chain Reaction --methodsen_US
dc.subject.meshPolymorphism, Single-Stranded Conformationalen_US
dc.subject.meshSensitivity and Specificityen_US
dc.titleInexpensive, rapid and convenient PCR-minigel SSCP protocol for polymorphisms and mutations analyses of LDL receptor gene.en_US
dc.typeJournal Articleen_US
dc.typeResearch Support, Non-U.S. Gov'ten_US
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