Niciura Simone Cristina Méo, Cruvinel Giovanna Gabrielle, Moraes Caroline Valério, Bressani Flavia Aline, Malagó Junior Wilson, Benavides Magda Vieira, de Chagas Ana Carolina Souza
Embrapa Pecuária Sudeste, Rodovia Washington Luiz, km 234, Fazenda Canchim, PO Box 339, São Carlos, SP, CEP 13560-970, Brazil.
Centro Universitário Central Paulista, Rua Miguel Petroni, 5111, São Carlos, SP, CEP 13563-470, Brazil.
Mol Biol Rep. 2018 Aug;45(4):651-656. doi: 10.1007/s11033-018-4206-8. Epub 2018 Jun 5.
Single nucleotide polymorphisms (SNPs) are the main type of variation in genome, enabling them to be associated with traits of economic importance in livestock. Genome-wide association studies (GWAS) have led to the discovery of SNPs associated with desirable traits in sheep. However, in these studies, SNPs are genotyped by high-throughput methods in genome scale, which are expensive and require sophisticated equipment and analysis methods. Therefore, the goal of this study was to develop a reliable, rapid, and inexpensive polymerase chain reaction (PCR)-based method to genotype a medium number of animals for a few candidate SNPs previously associated with desirable phenotypes in sheep by GWAS, using markers associated with gastrointestinal nematode resistance as a model. DNA extracted from white-blood cells of 150 sheep was submitted to PCR amplification followed by agarose gel electrophoresis and determination of banding pattern. Tetra-primer ARMS-PCR was successfully optimized after changes in annealing temperature; annealing and extension times; concentration of MgCl and DNA; ratios of inner, outer, forward and reverse primer; and addition of adjuvants, for genotyping the OAR2_14765360, OAR6_81718546, OAR11_62887032, and OAR12_69606944 SNPs in sheep. An extensive optimization of tetra-primer ARMS-PCR resulted in a suitable, simple, cost-effective PCR-based method of genotyping four SNP markers previously detected by chip arrays.
单核苷酸多态性（SNPs）是基因组变异的主要类型，这使得它们能够与家畜的经济重要性状相关联。全基因组关联研究（GWAS）已发现与绵羊优良性状相关的SNPs。然而，在这些研究中，SNPs是通过基因组规模的高通量方法进行基因分型的，这些方法成本高昂，需要精密的设备和分析方法。因此，本研究的目的是开发一种可靠、快速且廉价的基于聚合酶链反应（PCR）的方法，以对中等数量的动物进行基因分型，针对一些先前通过GWAS与绵羊优良表型相关的候选SNPs，使用与胃肠道线虫抗性相关的标记作为模型。从150只绵羊的白细胞中提取的DNA进行PCR扩增，随后进行琼脂糖凝胶电泳并确定条带模式。在对退火温度、退火和延伸时间、MgCl₂和DNA浓度、内引物、外引物、正向和反向引物的比例以及添加佐剂进行改变后，成功优化了四引物扩增受阻突变体系PCR（Tetra-primer ARMS-PCR），用于绵羊OAR2_14765360、OAR6_817185
