Department of Pathology, University of Utah, Salt Lake City, UT, USA.
BioMerieux Inc, Salt Lake City, UT, USA.
Mol Aspects Med. 2024 Jun;97:101268. doi: 10.1016/j.mam.2024.101268. Epub 2024 Mar 15.
Melting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time instruments with fluorescently labeled probes or dyes. Dyes monitor the entire PCR product, while probes focus on a specific locus within the amplicon. Advances in amplicon melting include high resolution instruments, saturating DNA dyes that better reveal multiple products, prediction programs for domain melting, barcode taxonomic identification, high speed microfluidic melting, and highly parallel digital melting. Most single base variants and small insertions or deletions can be genotyped by high resolution amplicon melting. High resolution melting also enables heterozygote scanning for any variant within a PCR product. A web application (uMelt, http://www.dna-utah.org) predicts amplicon melting curves with multiple domains, a useful tool for verifying intended products. Additional applications include methylation assessment, copy number determination and verification of sequence identity. When amplicon melting does not provide sufficient detail, unlabeled probes or snapback primers can be used instead of covalently labeled probes. DNA melting is a simple, inexpensive, and powerful tool with many research applications that is beginning to make its mark in clinical diagnostics.
熔化是 DNA 的基本性质，可以通过吸光度或荧光来监测。PCR 方便地产生足够的 DNA，可直接在实时仪器上用荧光标记的探针或染料进行监测。染料监测整个 PCR 产物，而探针则集中在扩增子内的特定位置。扩增子熔化技术的进步包括高分辨率仪器、饱和 DNA 染料，可更好地揭示多个产物、域熔化预测程序、条码分类鉴定、高速微流熔化和高度并行数字熔化。大多数单碱基变体和小的插入或缺失都可以通过高分辨率扩增子熔化进行基因分型。高分辨率熔化还可用于扫描 PCR 产物中任何变体的杂合子。一个网络应用程序（uMelt，http://www.dna-utah.org）可以预测具有多个域的扩增子熔化曲线，这是验证预期产物的有用工具。其他应用包括甲基化评估、拷贝数确定和序列同一性验证。当扩增子熔化不能提供足够的细节时，可以使用未标记的探针或回扫引物代替共价标记的探针。DNA 熔化是一种简单、廉价、功能强大的工具，具有许多研究应用，开始在临床诊断中崭露头角。
