样本数量和终质量浓度对DNA混合池PCR扩增质量和测序效果的影响
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摘要
为筛选适宜的DNA混合池法,采用两因素交叉分组设计,以混合样本数量(20、60和150个)和终质量浓度(50、100和150 ng/μL)为组合因子,分析不同混合池对PCR扩增质量及SNPs检出率的影响。结果表明:在同一混合样本数量下,以终质量浓度100 ng/μL建立的DNA混合池扩增的PCR产物纯度和OD_(260)/OD_(280)均优于50 ng/μL,但对测序结果无显著影响;以终质量浓度150 ng/μL建立的DNA混合池扩增的PCR产物杂带较多,不符合测序要求。在同一混合样本终质量浓度下,混合样本数越多,SNPs检出率越高。综上,从PCR扩增质量、SNPs检出率和试验成本来分析,以混合样本150个、终质量浓度100 ng/μL组合建立的DNA混合池效果最好。
To screen suitable method for DNA mixing pool, the experimental design of two-factor cross-sectional with mixed sample numbers(20, 60 and 150)and final mass concentration(50,100 and 150 ng/μL)were used to analyze the effect of different DNA mixing pools on PCR amplification quality and SNPs detection efficiency.The results indicated that under the same mixed sample numbers, the PCR products purity and OD_(260)/OD_(280) amplified by DNA mixing pool of 100 ng/μL final mass concentration were better than 50 ng/μL final mass concentration, and their sequencing results had no significant effect. PCR amplification product with DNA mixing pool of 150 ng/μL final mass concentration didn't meet sequencing requirements because it had more heterotopic bands. The more mixed sample numbers, the higher SNPs detection efficiency under the same final mass concentration. In conclusion, the DNA mixing pool for the combination of 150 mixed samples and 100 ng/μL final mass concentration was the best.
To screen suitable method for DNA mixing pool, the experimental design of two-factor cross-sectional with mixed sample numbers(20, 60 and 150)and final mass concentration(50,100 and 150 ng/μL)were used to analyze the effect of different DNA mixing pools on PCR amplification quality and SNPs detection efficiency.The results indicated that under the same mixed sample numbers, the PCR products purity and OD_(260)/OD_(280) amplified by DNA mixing pool of 100 ng/μL final mass concentration were better than 50 ng/μL final mass concentration, and their sequencing results had no significant effect. PCR amplification product with DNA mixing pool of 150 ng/μL final mass concentration didn't meet sequencing requirements because it had more heterotopic bands. The more mixed sample numbers, the higher SNPs detection efficiency under the same final mass concentration. In conclusion, the DNA mixing pool for the combination of 150 mixed samples and 100 ng/μL final mass concentration was the best.
引文
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[2]DARVASI A,SCOLLER M.Selective DNA pooling for determination of linkage between a molecular marker and a quantitative trait locus[J].Genetics,1994,138(4):1365-1373.
[3]刘文博,陈思睿,郑江霞,等.利用混合样本池法对鸡显性白羽基因PMEL17突变位点的检测[J].遗传(北京),2010,32(2):148-152.LIU W B,CHEN S R,ZHENG J X,et al.Detection of chicken dominant white gene PMEL17 mutation site by a pooling method[J].Hereditas(Beijing),2010,32(2):148-1527.
[4]刘果,谢耀坚,张党权,等.基因组DNA混合池技术在SSR引物筛选中的可靠性分析[J].分子植物育种,2018,16(3):903-913.LIU G,XIE Y J,ZHANG D Q,et al.Reliability analysis of genomic DNA mixing pool technology in SSR primer screening[J].Molecular Plant Breeding,2018,16(3):903-913.
[5]曹唱唱.混合测序实验设计及数据分析[D].南京:东南大学,2017.CAO CH CH.Pooled sequencing:experimental desing and data analysis[D].Nanjing:Southeast University,2017.
[6]高小莉.牦牛DGAT1基因多态性及其与乳品质性状关联研究[D].兰州:甘肃农业大学,2018.GAO X L.Polymorphisms of the DGAT1 gene and its association with milk quality traits in yak[D].Lanzhou:Gansu Agricultural University,2018.
[7]崔建勋,杜红丽,张细权.利用DNA池和测序技术快速筛查SNPs及估算基因频率[J].遗传学报,2005,32(4):372-377.CUI J X,DU H L,ZHANG X Q.Rapidly screening SNPs and estmiating allelic frequencies by DNA pooling and sequencing[J].Acta Genetica Sinica,2005,32(4):372-377.
[8]初芹,李东,侯诗宇,等.基于DNA池测序法筛选奶牛高信息量SNP标记的可行性[J].遗传,2014,36(7):691-696.CHU Q,LI D,HOU SH Y,et al.Direct sequencing of DNApooling for screening highly informative SNPs in dairy cattle[J].Hereditas,2014,36(7):691-696.
[9]J萨姆布鲁克,D W拉塞尔.分子克隆实验指南[M].黄培堂,朱厚础,张兆山,等译.第3版.北京:科学出版社,2002:611-618.SAMBROOK J,RUSSELL D W.Molecular Cloning:a Laboratory Manual[M].Translated by HUANG P T,ZHU HCH,ZHANG ZH SH,et al.3rd ed.Beijing:Science Press,2002:611-618.
[10]何柳,唐迅,胡永华.DNA混合分析技术的单体型频率估计方法[J].中南大学学报(医学版),2011,36(5):457-461.HE L,TANG X.HU Y H.Estimation of haplotypes based on DNA pooling[J].Journal of Central South University(Medical Sciences),2011,36(5):457-461.
[11]徐建峰,张艳丽,容维中,等.早胜牛及其杂交类群IGF-II基因多态性与肉用性状关联分析[J].西北农业学报,2016,25(7):955-959.XU J F,ZHANG Y L,RONG W ZH,et al.Association analysis between polymorphism of IGF-II gene in exon 2and beef performance traits in Zaosheng cattle and hybrid groups[J].Acta Agriculturae Boreali-occidentalis Sinica,2016,25(7):955-959.
[12]KOOHMARAIE M.KENT M P,SHACKELFORD S D,et al.Meat tenderness and muscle growth:is there any relationship[J].Meat Science,2002,62:345-352.
[13]PAGE B T,CASAS E,HEATON M P,et al.Evaluation of single-nucleotide polymorphisms in CAPN1for association with meat tenderness in cattle[J].Journal of Animal Science,2002,80(12):3077-3085.
[14]RAYNAUD P,TAYAT C,LAFORET M,et al.Four promoters direct expression of the calpastatin gene[J].Archives of Biochemistry and Biophysics,2005,437(1):69-77.
[15]COSTELLO S,DOHERTY E,TROY D J,et al.Association of polymorphisms in the calpainⅠ,calpainⅡand growth hormone genes with tenderness in bovine longissimusdor[J].Meat Science,2007,75(4):551-557.
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