The extraction and quantitative of DNA 许冰莹 教授 昆明医科大学法医学院

哪些样本可以提取DNA？The DNA can be extracted from which samples? 2

毛发 唾液（斑） 精液（斑） 血液（斑） 组织 指甲 牙齿 骨骼 组织（tissue）指甲（nail）牙齿（tooth）骨骼（bone）血液（blood）精液（semen）唾液（saliva）毛发（hair）

the type of sample 血液 20000~40000ng/ml 血斑（1cm2） 250~500 ng 精液 性交后阴道拭子 拔下头发 1~750 ng/根 自然脱落头发 1~12 ng/根 唾液（saliva） 1 000~10 000 ng/ml 口腔拭子 1 000~1 500 ng 尿液（urine） 1~20 ng/ml 骨骼 3~10 ng/mg 样品类型(the type of sample) 性交后阴道拭子 (Post-coital vaginal swabs)口腔拭子(oral swab)唾液（saliva）尿液（urine）DNA含量（the type of sample）

1. DNA的提取 概 述 各类样本处理 常用提取方法 概 述 常用提取方法 各类样本处理 概述summary 常用提取方法Commonly used extraction method 各类样品处理All kinds of sample processing

The extraction of DNA specimens 1.The separation of the biological samples and the abiotic components the extraction of DNA specimens 2.Extract the DNA from the cell and separate from cells of other components summary

How to obtain nuclear DNA? summary

The theory of the extraction nDNA biological cell SDS hypotension The theory of the extraction nDNA nuclear SDS heated to boil 核膜破裂，DNA从核内释放 protease K 水解DNA结合的蛋白质，DNA游离在溶液中 磁珠、硅胶magnetic beads、silica gel 酚、氯仿、乙醇phenol、chloroform、ethanol 杂质(impurity)去除，收集 DNA summary

Extraction of ideal DNA  （1）最大程度的去除样品DNA溶液中的RNA、蛋白质(protein)、多糖(polysaccharide)和脂类分子(lipid molecules)等成分； （2）尽可能的保持提取的DNA分子的完整性； （3）减少或去除可能对酶(enzyme)有抑制作用(inhibition)的物质，如有机溶剂(organic solvent)、去污剂(decontaminant)、金属离子(metal ion)、染料(dyestuff)及其他抑制DNA分析所用酶活性的物质。 summary

Chelex-100 method Commonly used extraction method Differential lysis Saturated phenol - chloro extraction method Chelex-100 method Differential lysis method Others: magnetic bead method

Saturated phenol - chloro extraction method sample digestion 裂解细胞膜核膜，消化核蛋白 去除蛋白质、多糖等杂质 phenol - chloro extraction DNA pellet dry

sample digestion SDS PrK TES

Protein precipitation (phenol/chloroform/polysaccharide） extraction aqueous phrase（DNA） Protein precipitation oganic phrase (phenol/chloroform/polysaccharide）

重复酚－氯仿抽提2～3次 注意：动作轻柔 避免皮肤粘膜接触试剂 小心吸取 加入 酚－氯仿 混匀 离心

DNA沉淀干燥 预冷的 70％乙醇 预冷的 无水乙醇 离心 3M NaCl 弃无水乙醇 干燥

ADVANTAGE： (1)A wide range of applications, suitable for all biological samples； (2)More effective against corruption, pollution, old samples and nails, bone and other materials； (3) DNA high purity, can be used for all DNA analysis。

DISADVANTAGE： The use of harmful chemical reagent, extraction process requires multiple centrifugation, may lead to more loss of DNA and may remain inhibited ingredients in the extract.

Chelex-100 method Commonly used extraction method Differential lysis Saturated phenol - chloro extraction method Chelex-100 method Differential lysis method Others: magnetic bead method

2.Chelex-100 extraction method Chelex-100是一种化学螯合树脂(Chelating resin)，由苯乙烯、二乙烯苯共聚体组成，含有成对的亚氨基二乙酸盐离子，对金属离子有很强的螯合作用。 5％Chelex-100

Chelex-100 extraction steps 样本 Chelex-100 悬浮液 Chelex-100颗粒

Chelex-100 extraction steps 振荡 振荡 56℃ 0.5～12h 100℃ 10min DNA在上 清液中 振荡：shake 离心：Centrifugal DNA在上 清液中：DNA in the supernatant 蛋白质沉淀、与金属离子结合颗粒：Protein precipitation, granule combined with metal ion 离心 蛋白质沉淀、与金属离子结合颗粒

注 意（attention） (1)如Chelex-100提取的DNA要放置一段时间才分析，因Chelex-100随时间延长会释放出以前结合的抑制剂或金属离子，而抑制PCR反应，故应把上清液与Chelex-100颗粒分开，保存在-20℃。 (2) Chelex-100结合效果依赖于悬浮液pH值，pH值在10.0～11.0为好。 (3) Chelex-100能螯合镁离子，PCR反应中不能混有Chelex-100树脂。

Less amount of samples, suitable for extracting trace materials DNA; ADVANTAGE: simple and quick; Less amount of samples, suitable for extracting trace materials DNA; Extraction is always in the same tube, can reduce the loss of DNA； The degradation of DNA can be prevented by Chelex-100. 优点: 简便快速；simple and quick 检材用量少，适合微量检材的DNA提取；Less amount of samples, suitable for extracting trace materials DNA 提取始终在同一试管中，可减少DNA的损失；Extraction is always in the same tube, can reduce the loss of DNA Chelex-100本身可防止DNA的降解。Degradation of Chelex-100 can prevent DNA 缺点: (1)提取的DNA产物中不可避免地残留部分杂质;Inevitably some residual impurities in DNA product extraction (2) Chelex-100处理后得到的是单链DNA，不能用于RFLP分析。Get after the treatment of Chelex-100 is single-stranded DNA, cannot be used for RFLP analysis

DISADVANTAGE: (1)Inevitably some residual impurities in DNA product extraction; (2)Get after the treatment of Chelex-100 is single-stranded DNA, cannot be used for RFLP analysis.

Blood and blood stain samples

The sediment containing the nucleus blood Centrifugal, abandoned the plasma RBC Low permeability, breaking cell Hemolytic solution containing the nucleus Washing, centrifugation, except Hb The sediment containing the nucleus Washing, centrifugation, except Hb Extraction of DNA phenol-chloroform method or Chelex-100 method Samples dissolved in Hb Chopped, soaked in dH2O blood stain

Semen stain and mixed plaque samples 精斑、混合斑检材 剪碎，dH2O浸泡 去载体，离心 沉渣 液体 差异裂解法提取 抗P30 血型测定 涂片 找精子

Other samples 酚－氯仿法或Chelex-100法提取DNA salivary stain 毛发 costal cartilage 牙齿 剪碎 取毛囊 切薄片 牙髓 酚－氯仿法或Chelex-100法提取DNA

2.Quantitative detection of DNA Ethidium bromide fluorescence detection method ultraviolet spectrophotometry Dot blot hybridization assay Real-time fluorescent quantitative PCR technique

Ethidium bromide fluorescence detection method 将提取的 DNA 液与一系列已知浓度的DNA样品液在同一琼脂糖凝胶上加样电泳，然后用溴乙锭(EB)染色。溴乙锭分子能够嵌入DNA双链分子间，在紫外光激发下显示红色荧光，荧光强度与DNA的浓度成正比。 EB荧光测定法灵敏度高，可以检测出1～5ngDNA。 将提取的 DNA 液与一系列已知浓度的DNA样品液在同一琼脂糖凝胶上加样电泳，然后用溴乙锭(EB)染色。溴乙锭分子能够嵌入DNA双链分子间，在紫外光激发下显示红色荧光，荧光强度与DNA的浓度成正比。 EB荧光测定法灵敏度高，可以检测出1～5ngDNA。 The DNA sample liquid liquid extraction of DNA and a series of known concentration in the same agarose gel electrophoresis and kind，Then using ethidium bromide (EB) staining. Ethidium bromide molecules can be inserted into the DNA double chain molecule,Show red fluorescence under ultraviolet excitation, fluorescence intensity and DNA concentration is proportional to the. EB fluorescence assay with high sensitivity, can detect 1 ~ 5ngDNA。

Analyzing DNA samples in a Classroom Lab A B C D E Ladder Analysis of samples: Barley (A): This sample is fine Corn (B): This sample is fine Oat (C) : This sample is fine Rice (D) : This sample is fine Wheat (E): This sample has severe degradation, can work for PCR but should re-extract

Ultraviolet spectrophotometric method and principle basic group 腺嘌呤 胞嘧啶 鸟嘌呤 胸腺嘧啶 A C G T Maximum Absorbance（nm） 260.5 267 267 264.5 核酸的最大吸收波长为260nm， 在260nm紫外光下，1 OD值相当于50μg/ml浓度的双链DNA，40μg/ml浓度的单链DNA溶液。

将提取的样本DNA溶解于0.1mol/LNaOH 液中，经分光光度计测定OD260 值，按照公式计算样本DNA的含量： 样本DNA浓度(μg/ml)＝OD260×40μg/ml×稀释倍数 蛋白质最大吸光波长是280nm，同时测定样本DNA液OD260值和OD280值，计算OD260/OD280 比值，如果比值为 1.8－2.1，样本DNA比较纯；如果比值小于1.6，样本液中含有过多的蛋白质杂质。