SNP genotyping through the melting analysis of unlabelled oligonucleotide applied on dilute PCR amplicon |
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Authors: | Jeong Sangkyun Yu Hyunjoo Lee Yeongshin Kim Jong Yeol |
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Institution: | Division of Constitutional Medicine Research, Korea Institute of Oriental Medicine, 483 Exporo, Yuseong-gu, Daejon 305-811, Republic of Korea |
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Abstract: | Adaptation of DNA melting analysis for polymorphic single nucleotides (SNPs) genotyping using an unlabeled oligonucleotide probe for polymorphic DNAs under the presence of fluorescent DNA binding dye necessitates a reaction condition where the probe efficiently associates with a target strand that is PCR amplified. We present experimental evidence that application of an unlabeled probe to a dilute PCR amplicon provides a condition such that the fluorescent signals gained subsequently by probe melting are sufficient to discriminate allelic identities. This approach is best exploited by adapting the multiplexing PCR technique in order to cover multiple SNPs for given samples. 3′-end modification of the probe is unnecessary as the amplicon dilution step provides a way of inactivating the polymerase through divalent cation chelation. With the use of low-cost reagents and ordinary laboratory equipment, this method offers a rapid, simple and cost-efficient way of SNP genotyping. |
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Keywords: | SNP single nucleotide polymorphism GWAS genome-wide association study UOP unlabelled oligonucleotide probe bps base pairs TM melting temperature |
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