核酸检测技术

1、第2章 核酸检测技术,核酸检测技术 直接对动植物有害生物基因序列和结构进行测定。 核酸的分离纯化 PCR技术 核酸杂交技术,第1节 核酸的分离纯化,核酸的分离纯化的原则,保持核酸一级结构的完整性 提高核酸制品的纯度,技术路线的设计,破碎细胞的方法,机械 （匀浆、超声破、研磨等）,非机械（化学处理、生化法）,沉淀是浓缩核酸最常用且高效的方法。,核酸浓度检测与完整性测定方法,浓度测定 紫外分光光度法 260nm 荧光光度法 EB荧光,完整性测定 琼脂糖凝胶电泳法：以EB为示踪剂的核酸凝胶电泳结果为依据。 基因组DNA片段如发生降解，电泳图呈拖尾状； 完整的或降解很少的总RNA电泳图谱中，三条带的荧

2、光强度积分应呈特定的比值,问题一：DNA样品不纯 抑制后续酶解和PCR反应,DNA提取常见问题,DNA提取常见问题,问题二： DNA降解,DNA提取常见问题,问题三： DNA提取量少,真菌,昆虫,第2节 PCR扩增技术,PCR, a DNA photocopier,PCR技术简史,DNA的复制 核酸体外扩增的设想 聚合酶链反应的发明,1985年，美国PE-Cetus公司的Mullis等人发明了聚合酶链反应（PCR） 最初采用E-coli DNA聚合酶进行PCR，由于该酶不耐热，使这一过程耗时，费力，且易出错 耐热DNA聚合酶的应用使得PCR能高效率的进行，随后PE-Cetus公司推出了第一台P

3、CR自动化热循环仪 1993年，Mullis等因此项技术获诺贝尔化学奖,PCR技术简史,DNA的复制 核酸体外扩增的设想 聚合酶链反应的发明,Kary B. Mullis（1944）,1989年美国Science杂志列PCR 为十余项重大科学发明之首,比喻1989年为PCR爆炸年,Mullis荣获1993年度诺贝尔化学奖。,1. PCR的基础,Polymerase,Chain,Reaction,PCR,The only enzyme used in this reaction is DNA polymerase.,Polymerase,Chain,Reaction,The products o

4、f the first reaction become substrates of the following one, and so on.,PCR,Polymerase,Chain,Reaction,PCR,Components： Thermostable DNA Polymerase， Target DNA，Pair of Primers， dNTPs， Mg+ ions， Buffer solution,Denaturation：The target DNA (template) is separated into two stands by heating to 95 Primer

5、annealing: The temperature is reduced to around 55 to allow the primers to anneal. Polymerization (elongation, extension): The temperature is increased to 72 for optimal polymerization step which uses up dNTPs and required Mg2+.,The PCR cycle: Three different steps in each PCR cycle,PCR的扩增？,How does

6、 PCR work,How does PCR work,The third cycle,The fourth cycle,2. PCR的类型,多重PCR（multiplex PCR） 巢式PCR（nested PCR） 定量PCR（quantitative PCR） 不对称PCR（asymmetric PCR） 反向PCR（inverse PCR） 逆转录PCR（reverse transcription PCR） Overlap PCR ,PCR using several primer pairs SIMULTANEOUSLY Typically generates a product b

7、and for each primer pair,多重PCR（multiplex PCR）:What,2.1 多重PCR（multiplex PCR）,Multiplex PCR: Why,Detect several genes at once eg. transgenic plant screen Internal controls VERY important Tells you how well the PCR reaction worked Reduces “false negatives” Reduces “false positives”,多重PCR（multiplex PCR）

8、,Multiplex PCR: How,Same as regular PCR Care in primer design Much greater chance of primer-dimers Much greater chance of artifacts Annealing temperatures must be close,多重PCR（multiplex PCR）,A Typical Multiplex PCR Reaction,Sterile Water 34.0 ul 10X PCR Buffer 5.0 ul MgCl2 (50mM) 2.5 ul dNTPs (10mM e

9、ach) 1.0 ul Primer1FWD 1.0 ul Primer1REV 1.0 ul Primer2FWD 1.0 ul Primer2REV 1.0 ul Primer3FWD 1.0 ul Primer3REV 1.0 ul DNA Polymerase 0.5 ul DNA Template 1.0 ul Total Volume 50.0 ul,多重PCR（multiplex PCR）,Multiplex PCR: Example,Three primer pairs Control, resistance, and trait genes Control gene frag

10、ment is largest and (almost) faintest Trait gene is smallest and brightest,Resistance Gene,Trait Gene,Control Gene,Primers,多重PCR（multiplex PCR）,Multiplex PCR: Example,Three primer pairs,Resistance Gene,Trait Gene,Which are transgenic?,Control Gene,Primers,多重PCR（multiplex PCR）,Nested Multiplex PCR,2n

11、d Stage PCR,Dilute 100 fold Between 1st dependent on mutation within recognition site of restriction enzyme Former used with southern blot experiments Even as many restriction enzymes are known, some mutation sites do not correspond to any,- Rare endonucleases are difficult to work with, and often o

12、f a poor quality,Restriction fragment length polymorphism (RFLP),Restriction fragment length polymorphism (RFLP),Modified method: Diagnostic restriction site introduced artificially by purposedly ismatched PCR primer,140,PCR based methods,Random Amplified Polymorphic DNA (RAPD),RAPD is often used to

13、 show relatedness among DNA populations. In this procedure arbitrary (random) primers are used during PCR to produce a fingerprint of the DNA. A single primer is used which must anneal in 2 places on the DNA template and region between the primers will be amplified.,The primers (8-10nt) are likely t

14、o anneal in many places on the template DNA and will produce a variety of sizes of amplified products. Amplified products are separated by agarose gel electrophoresis and visualized. If the samples have similar genetic make up then the pattern of bands on the gel will be similar and vice versa. This proce