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长牡蛎(Crassostrea gigas)基因组具有高度的多态性。因此,导致传统的高分辨率熔解曲线(HRM)方法在长牡蛎基因组SNP分型验证中表现出很低的效率,并且传统HRM方法花费比较高。本文提出一种经过改良的适合高度多态性基因组SNP验证的HRM方法。这种two-step HRM方法在传统的HRM方法基础上进行了许多改进;利用这种改进的方法,使用一对引物即可准确完成一个特定SNP位点基因型的确定和区分,并且相应的花费较传统方法大大降低。本实验中共使用56组引物对长牡蛎SNP进行验证,有8组引物因为特异性较差在引物筛选中被排除。最终有30组引物成功验证SNP,成功率约为62.5%。超过63%的SNP位点为转换类型(C/T:30%,A/G:33.3%),只有大约36.7%的SNP位点为颠换类型(A/C:10%,G/T:10%,A/T:10%,C/G:6.7%)。随即选取5组引物的扩增产物进行Sanger测序,通过序列对比后获得与该方法完全相同的分型结果,这也证明了本方法的准确性。以上数据表明本文提出的two-step HRM方法是一种可使用于高多态性牡蛎基因组的高效、经济并且有高通量潜力的SNP验证方法。
The genome of Crassostrea gigas is highly polymorphic. As a result, the traditional high-resolution melting curve (HRM) approach has shown to be inefficient at prototyping SNP genomic SNPs and the traditional HRM method is expensive. This paper presents an improved HRM method suitable for SNP verification of highly polymorphic genomes. The two-step HRM method has made many improvements based on the traditional HRM method. By using the improved method, a pair of primers can accurately determine and distinguish genotypes of a specific SNP locus, and the corresponding cost Compared with the traditional method greatly reduced. In this experiment, a total of 56 primers were used to verify the SNP of long oyster, 8 primers were excluded in the primer screening because of their poor specificity. Finally, 30 sets of primers successfully verified SNP, the success rate was about 62.5%. Over 63% of the SNP loci were CTC (30% for C / T, 33.3% for A / G) and only 36.7% for SNP sites were transversions (A / C: 10%, G / T: 10%, A / T: 10%, C / G: 6.7%). Then, the amplification products of the five primer sets were selected for Sanger sequencing, and the exact same typing result was obtained by sequence comparison, which proves the accuracy of the method. The above data indicate that the proposed two-step HRM method is an efficient, economical and high-throughput SNP validation method that can be applied to the highly polymorphic oyster genome.