Division of Constitutional Medicine Research, Korea Institute of Oriental Medicine, 483 Exporo, Yuseong-gu, Daejon 305-811, Republic of Korea.
J Biotechnol. 2011 Jul 20;154(4):321-5. doi: 10.1016/j.jbiotec.2011.05.017. Epub 2011 Jun 12.
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.
利用荧光 DNA 结合染料在存在条件下对多态性单核苷酸 (SNPs) 进行 DNA 熔解分析的适配，需要一种反应条件，使探针能够有效地与 PCR 扩增的靶链结合。我们提供了实验证据，表明将未标记的探针应用于稀释的 PCR 扩增子可以提供一种条件，使得随后通过探针熔解获得的荧光信号足以区分等位基因身份。通过适应多重 PCR 技术以覆盖给定样品中的多个 SNPs，可以最好地利用这种方法。探针的 3'-端修饰是不必要的，因为扩增子稀释步骤通过二价阳离子螯合提供了一种使聚合酶失活的方法。使用低成本试剂和普通实验室设备，这种方法提供了一种快速、简单和经济高效的 SNP 基因分型方法。
