Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, 430062, People's Republic of China.
Institute of Vegetables and Flowers of the Chinese Academy of Agricultural Sciences, Beijing, 100081, People's Republic of China.
Plant Methods. 2012 Aug 24;8(1):34. doi: 10.1186/1746-4811-8-34.
Although Single Nucleotide Polymorphism (SNP) marker is an invaluable tool for positional cloning, association study and evolutionary analysis, low SNP detection efficiency by Allele-Specific PCR (AS-PCR) still restricts its application as molecular marker like other markers such as Simple Sequence Repeat (SSR). To overcome this problem, primers with a single nucleotide artificial mismatch introduced within the three bases closest to the 3'end (SNP site) have been used in AS-PCR. However, for one SNP site, nine possible mismatches can be generated among the three bases and how to select the right one to increase primer specificity is still a challenge.
In this study, different from the previous reports which used a limited quantity of primers randomly (several or dozen pairs), we systematically investigated the effects of mismatch base pairs, mismatch sites and SNP types on primer specificity with 2071 primer pairs, which were designed based on SNPs from Brassica oleracea 01-88 and 02-12. According to the statistical results, we (1) found that the primers designed with SNP (A/T), in which the mismatch (CA) in the 3rd nucleotide from the 3' end, had the highest allele-specificity (81.9%). This information could be used when designing primers from a large quantity of SNP sites; (2) performed the primer design principle which forms the one and only best primer for every SNP type. This is never reported in previous studies. Additionally, we further identified its availability in rapeseed (Brassica napus L.) and sesame (Sesamum indicum). High polymorphism percent (75%) of the designed primers indicated it is a general method and can be applied in other species.
The method provided in this study can generate primers more effectively for every SNP site compared to other AS-PCR primer design methods. The high allele-specific efficiency of the SNP primer allows the feasibility for low- to moderate- throughput SNP analyses and is much suitable for gene mapping, map-based cloning, and marker-assisted selection in crops.
虽然单核苷酸多态性（SNP）标记是定位克隆、关联研究和进化分析的宝贵工具，但等位基因特异性 PCR（AS-PCR）的 SNP 检测效率低仍然限制了它作为分子标记的应用，就像简单序列重复（SSR）等其他标记一样。为了克服这个问题，在最靠近 3'端（SNP 位点）的三个碱基内引入单个核苷酸人工错配的引物已被用于 AS-PCR。然而，对于一个 SNP 位点，在三个碱基中可以产生九种可能的错配，如何选择正确的错配来提高引物特异性仍然是一个挑战。
在这项研究中，与之前使用有限数量的引物随机（几对或十几对）的报告不同，我们系统地研究了错配碱基对、错配位点和 SNP 类型对基于 Brassica oleracea 01-88 和 02-12 的 SNP 设计的 2071 对引物特异性的影响。根据统计结果，我们 (1) 发现，在 3'端第三个核苷酸处的错配 (CA) 为 SNP (A/T) 设计的引物具有最高的等位基因特异性（81.9%）。这一信息可用于设计大量 SNP 位点的引物；(2) 提出了一种引物设计原则，为每种 SNP 类型形成唯一最佳引物。这在以前的研究中从未报道过。此外，我们还进一步确定了它在油菜（Brassica napus L.）和芝麻（Sesamum indicum）中的可用性。设计引物的高多态性百分比（75%）表明这是一种通用方法，可应用于其他物种。
与其他 AS-PCR 引物设计方法相比，本研究提供的方法可以更有效地为每个 SNP 位点生成引物。SNP 引物的高等位基因特异性效率使得低至中通量 SNP 分析成为可能，非常适合作物的基因作图、基于图谱的克隆和标记辅助选择。
