454技术培训.ppt

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2018-03-23 发布于河南
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454技术培训.ppt

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454技术培训

300-500 1000 000 400 共勉 454 技术培训上海美吉生物医药科技有限公司焦磷酸测序原理焦磷酸测序（Pyrosequencing）技术是新一代DNA序列分析技术，由4种酶催化的同一反应体系中的酶级联化学发光反应，在每一轮测序反应中，只加入一种dNTP,若该dNTP与模板配对，聚合酶就可以将其掺入到引物链中并释放出等摩尔数的焦磷酸基团（PPi）。 PPi可最终转化为可见光信号，并由PyrogramTM转化为一个峰值。每个峰值的高度与反应中掺入的核苷酸数目成正比。然后加入下一种dNTP,继续DNA链的合成。焦磷酸测序原理 Three hundred to five hundred bases three hundred to five hundred bases one million reads three hundred to five hundred bases one million reads Four hundred million high quality bases in less than ten hours P P T T T T Small Fragment Removal T T Small Fragment Removal 454 Library Library QC Average fragment length Between 600 bp and 900 bp Lower size cut-off 10% below 350 bp Library Quantitation 1 no dilution 2.50 × 109 2 2/3rd dilution of tube 1 1.67 × 109 3 2/3rd dilution of tube 2 1.11 × 109 4 2/3rd dilution of tube 3 7.41 × 108 5 2/3rd dilution of tube 4 4.94 × 108 6 2/3rd dilution of tube 5 3.29 × 108 7 2/3rd dilution of tube 6 2.19 × 108 8 2/3rd dilution of tube 7 1.46 × 108 Library Quantitation Denature Denature Emulsification Denature Emulsification Distribution Denature Emulsification Distribution Amplification Denature Emulsification Distribution Amplification Isolation and Enrichment of DNA Positive Beads Denature Emulsification Distribution Amplification Isolation and Enrichment of DNA Positive Beads Bio Bio Bio Bio Bio Bio Bio Denature Emulsification Distribution Amplification Isolation and Enrichment of DNA Positive Beads Bio Bio Bio Bio Bio Bio Bio Denature Emulsification Distribution Amplification Isolation and Enrichment of DNA Positive Beads Bio Bio Bio Bio Bio Bio Bio Denature Emulsification Distribution Amplification Isolation and Enrichment of DNA Positive Beads Bio Bio Bio Bio Bio Bio Bio Denature Emulsification Distribution Amplification Isolation and Enrichment of DNA Positive Beads Bio Bio Bio Bio Bio Bio Bio Denature Emulsification Distribution Amplification Isolation and Enrichment of DNA Positive Beads Bio Bio Bio Bio Bio Bio Bio