Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes e Alto Douro (IBB/CGB-UTAD), Quinta de Prados, Vila Real, 5001-801, Portugal.
Mol Ecol Resour. 2013 May;13(3):473-83. doi: 10.1111/1755-0998.12081. Epub 2013 Feb 25.
High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex-specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.
高分辨率熔解（HRM）分析是一种非常有吸引力和灵活的高级 PCR 后方法，具有基于 PCR 产物特异性熔解曲线检测的简单、快速和经济高效的基因型分析的高灵敏度/特异性。新一代实时 PCR 系统，以及改进的饱和 DNA 结合染料，能够在定量 PCR 后直接获取 HRM 数据。熔解行为特别受扩增子的长度、核苷酸序列和 GC 含量的影响。该方法在人类遗传学、生殖生物学、微生物学和生态学/野生种群保护等多个研究领域迅速扩展。在这里，我们基于 CHD1 基因性别特异性片段的扩增，开发了一种成功的用于鸟类性别鉴定的 HRM 方案。该熔解曲线模式允许对 14 种鸟类的 111 个样本（拔下的羽毛、肌肉组织、血液和口腔上皮细胞）进行有效的性别分化。此外，我们对 CHD1 基因扩增区域进行了测序，并证明了该策略对于不同大小差异（2bp 到 44bp）的各种扩增子（CHD1Z 和 CHD1W）的基因型鉴别具有实用性。所建立的方法学清楚地揭示了简单 HRM 测定法用于准确区分研究物种的性别（例如管内系统、高灵敏度和快速性）的优势。该方法的要求、优势和局限性为其在鸟类分子性别鉴定领域的应用提供了一个简单的指南。与以前的实时 PCR 方法相比，HRM 分析具有更高的灵敏度和分辨率，使其成为改进鸟类性别鉴定的先进分子方法的优秀方法。
