DNA池结合DHPLC和直接测序技术在江豚SNPs检测中的应用

Application of DNA pooling in combination with DHPLC and direct sequencing in SNPs detection of the finless porpoise(Neophocaena phocaenoides)

选取江豚基因组中的2个已知单核苷酸多态性（single nucleotide polymorphisms，SNPs）位点，通过PCR扩增，将PCR产物按基因频率不同制备成0～50％的11个DNA池（DNAp001），用于变性高效液相色谱（denaturing high performance liquid chromatography，DHPLC）和直接测序分析，以探讨DNA池中基因频率的最低要求。结果显示，当稀有等位基因的基因频率不少于5％时可在DHPLC检测过程中明显分辨；而利用DNA池进行直接测序时的基因频率则需达到10％。这提示，为保证DHPLC分析的准确性和可靠性，制备DNA池时等摩尔DNA混合的个体数最好不超过10个。DNA池结合DHPLC技术的高效性与准确性可在大规模的SNPs位点筛选中发挥作用。

Denaturing high performance liquid chromatography （ DHPLC ） and direct sequencing in combination with DNA pooling were used as possible strategies for discussing the least rare allele frequency of DNA pools for SNPs detection in the finless porpoise （ Neophocaena phocaenoides ）. To evaluate the utility and sensitivity of this approach, we constructed DNA pools comprised of 20 previously genotyped individuals with a frequency representation of 0% - 50% for the variant allele. The SNPs can be detected out at a frequency that no less than 5% when using DNA pooling and DHPLC. In contrast, fluorescent sequencing detected variants in the same pools only if the frequency of the less common allele was at least 10%. We conclude that the sample number of DNA pooling for DHPLC analysis should be no more than ten to ensure the efficiency and accuracy of SNPs discovery and screening. The high efficiency and accuracy of the present methods will make them effective in large-scale SNPs screening.