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09월 04일 17시 이후 : 초록수정 불가능, 일정확인 및 검색만 가능
제114회 대한화학회 학술발표회, 총회 및 기기전시회 안내
Detection of single nucleotide polymorphisms with DNAzyme and graphene oxide
2014년 9월 3일 15시 48분 13초
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10월 15일 (수요일) 16:00~19:00
건국대학교 생명공학과, Korea
Single nucleotide polymorphisms (SNPs) are caused by DNA sequences containing a single different nucleotide, which are often related with disease and drug efficiency to each individual. Therefore, a method of SNP detection is needed for the diagnosis of SNP-related diseases and the prescription of personalized medicine. In this study, we report a novel assay for detection of SNPs using graphene oxide (GO), DNAzyme (Dz), and fluorescent labeled DNA (F-DNA). GO has a high affinity to single strand nucleic acid with a fluorescence quenching near the GO surface, whereas it shows a weak affinity for the double stranded nucleic acids. Dz is a catalytic oligodeoxyribonucleotide that can cleave phosphodiester bonds in the target RNA. As a model system, we used a fragment of ABL RNA (45 mer) containing a point mutation (T315I) in ABL gene of BCR-ABL mutant chimeric gene that causes chronic myeloid leukemia (CML). The T315I point mutation results in drug (imatinib mesylate) resistance with no other alternative regimens. In our SNP detection system, Dz specifically cleaved T315I mutant ABL RNA and generated pieces of RNA. RNA/F-DNA duplexes were then formed by adding F-DNA that can anneal to a cleaved product of T315I mutant RNA. GO was then added to the reaction solution, and the fluorescence of these RNA/F-DNA duplexes generated from the T315I mutant RNA was not quenched by GO. However, the ABL RNA lacking the SNP (i.e. wild type ABL RNA) was not cleaved by Dz, and RNA/F-DNA duplexes were subsequently formed as partial duplexes with single stranded portions. The fluorescence of the partial duplex RNA/F-DNA was adsorbed onto GO with significant fluorescence quenching. We confirmed appreciable fluorescence differences between T315I mutant RNA and ABL wild type RNA, using the fluorescence quenching GO-based SNP detection, which will be applicable for the detection of SNPs in RNA with simplicity, sensitivity, and selectivity.
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