课件下载地址. Introduction. Molecular Biology and Public Health ( 分子生物学与公共卫生 ) 7.DNA recombination (DNA 重组概念 )

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1 课件下载地址 厦门大学公共卫生学院主页 学院概况 学科建设 病原微生物与抗感染治疗课题组 网页下方的课件下载 :MBPH1-10 Molecular Biology and Public Health ( 分子生物学与公共卫生 ) DNA/RNA manipulation 1 (DNA/RNA 操作 1) Introduction The methods depend upon, and are developed from, an understanding of the properties of biological macromolecules themselves Nucleic acids 1.Isolation/Extraction( 提取 ) 2.Electrophoresis( 电泳 ) 3.Restriction( 限制性酶切 ) 4.Hybridization( 杂交 ) 5.PCR( 聚合酶链式反应 ) 6.Genome sequence & analysis( 基因组测序 & 分析 ) 7.DNA recombination (DNA 重组概念 ) 8.DNA recombination technology and cloning( DNA 重组和克隆技术 )

2 1.DNA / RNA isolation Phenol chloroform extraction This method may take longer than a column-based system such as the silica-based purification, but has higher purity. Column methods also shear large DNA fragments, which may or may not be a problem depending on downstream applications. the advantage of high recovery of RNA: an RNA column is typically unsuitable for purification of short (<200 nucleotides) RNA species, such as mirna and trna. Reagents Phenol ( 苯酚 ): The phenol used for biochemistry comes as a watersaturated solution with Tris buffer, as a Tris-buffered 50% phenol, 50% chloroform solution, or as a Tris-buffered 50% phenol, 48% chloroform, 2% isoamyl alcohol solution (sometimes called "25:24:1"). Phenol is naturally somewhat water-soluble, and gives a fuzzy interface, which is sharpened by the presence of chloroform, and the isoamyl alcohol reduces foaming. Most solutions also have an antioxidant, as oxidized phenol damages the nucleic acids. For RNA purification, the ph is kept at around 4, which retains RNA in the aqueous phase preferentially. For DNA purification, the ph is usually near 7, at which point all nucleic acids are found in the aqueous phase. Chloroform( 氯仿 ): Chloroform is stabilized with small quantities of amylene or ethanol, because exposure of pure chloroform to oxygen and ultraviolet light produces phosgene gas. Some chloroform solutions come as pre-made a 96% chloroform, 4% isoamyl alcohol mixture that can be mixed with an equal volume of phenol to obtain the 25:24:1 solution. Isoamyl alcohol( 异戊醇 ): Isoamyl alcohol may reduce foaming and ensure inactivation of RNases. How it works? Phase separation by centrifugation of a mixture of the aqueous sample and a solution containing water-saturated phenol and chloroform, resulting in an upper aqueous phase and a lower organic phase (mainly phenol) Guanidinium thiocyanate, a chaotropic agent, is sometimes added to the organic phase to aid in the denaturation of proteins (such as those that strongly bind nucleic acids or those that degrade RNA). The nucleic acids (RNA and/or DNA) partition into the aqueous phase, while denatured protein partitions into the organic phase. The ph of the mixture determines which nucleic acids get purified. Under acidic conditions (ph 4-6): DNA partitions into the organic phase while RNA remains in the aqueous phase. Under neutral conditions (ph 7-8): both DNA and RNA partition into the aqueous phase. In a last step, the nucleic acids are recovered from the aqueous phase by precipitation with 2-propanol or ethanol. 2.Electrophoresis through a Gel separate DNA and RNA molecules according to size Principle: Linear DNA molecules migrate through the gel toward the positive pole with different rates when subject to an electrical field. The DNA molecules can be visualized by staining the gel with fluorescent dyes, such as ethidium Bromide. 8

3 Electrophoresis DNA and RNA molecules are negatively charged, thus move in the gel matrix toward the positive pole (+) Linear DNA molecules are separated according to size The mobility of circular DNA molecules is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled> linear> nicked or relaxed Some fundamental steps of electrophoresis 10 DNA separation by gel electrophoresis Electrophoresis Gel matrix ( 胶支持物 ) Gel matrix ( 胶支持物 ) is an inserted, jello-like porous material that supports and allows macromolecules to move through. large moderate small After electrophoresis

4 Electrophoresis To separate DNA of different size ranges Small size range of DNA: use polyacrylamide Large size range of DNA: use agarose gel Very large DNA (>30-50kb): use pulsed-field gel electrophoresis Polyacrylamide ( 聚丙烯酰胺 ): (1)has high resolving capability, and can resolve DNA that differ from each other as little as a single base pair/nucleotide. (2)but can only separate DNA over a narrow size range (1 to a few hundred bp). Electrophoresis Electrophoresis Electrophoresis Agarose ( 琼脂糖 ): (1)much less resolving power than polyacrylamide, (2)but can separate DNA molecules of up to tens of kb 4 kb 3 kb 2 kb 1 kb 0.5 kb Pulsed-field gel electrophoresis (PFGE) (1)The electric field is applied in pulses that are oriented orthogonally ( 直角地 ) to each other. (2)Separate DNA molecules according to their molecule weight, as well as to their shape and topological properties. (3)Can effectively separate DNA molecules over kb and up to several Mb in length.

5 Electrophoresis pulsed-field gel electrophoresis Switching between two orientations: the larger the DNA is, the longer it takes to reorient Electrophoresis Electrophoresis is also used to separate RNAs (1)RNA has a uniform negative charge as DNA does. (2)RNA is single-stranded and has extensive secondary and tertiary structure, which significantly influences its electrophoretic mobility. (3)RNA can be treated with reagent such as glyoxal ( 乙二醛 ) to prevent RNA base pairing, so that its mobility correlates with the molecular weight. Restriction digestion Nucleic acid 3.Restriction endonucleases cleave DNA molecules at particular sites Why use endonucleases? --To make large DNA molecules break into manageable pieces (fragments) 19

6 Restriction digestion Restriction endonucleases (RE)( ( 限制性内切酶 ) are the nucleases that cleave DNA at particular sites by the recognition of specific sequences. RE used in molecular biology typically recognize ( 识别 ) short (4-8bp) target sequences that are usually palindromic ( 回文结构 ), and cut ( 切割 ) at a defined sequence within those sequences. e.g. Eco RI 5.GAATTC..3.CTTAAG. Restriction digestion To name a restriction endonuclease: e.g. EcoRI Escherichia coli Genus Species R13 strain the 1 st such enzyme found Restriction digestion How to estimate the frequency of the RE in a DNA molecule or genome? The random occurrence of the hexameric ( 六核苷酸的 ) sequence: 1/4096 (4-6 =1/4 6 ) Randomly (The largest fragment) (The smallest fragment) Consider a linear DNA molecule with 6 copies of GAATTC: it will be cut into 7 fragments which could be separated in the gel electrophoresis by size digestionof a DNA fragment with endonuclease Eco RI

7 Restriction digestion Endonucleases are used to make restriction map: e.g. the combination of EcoRI + HindIII Allows different regions of one molecule to be isolate and a given molecule to be identified A given molecule will generate a characteristic series of patterns when digested with a set of different enzymes (1) Restriction enzymes differ in the recognition specificity: target sites are different. (2) Restriction enzymes differ in the length they recognized, and thus the frequencies differ. (3) Restriction enzymes differ in the nature of the DNA ends they generate: blunt/flush ends ( 平末端 ), sticky/staggered ends ( 粘性末端 ). (4) Restriction enzymes differ in the cleavage activity. Restriction digestion Restriction digestion blunt ends The 5 protruding ends are said to be sticky because they readily anneal through base-pairing to DNA molecules cut with the same enzyme sticky ends Reanneal with its complementary strand or other strands with the same cut recognition sequences and cut sites of various endonucleases

8 RFLP (Restriction fragment length polymorphism, 限制性片段长度多态性 ) RLFP 分析法 由于 DNA 多态性, 取代的碱基正好位于某一限制酶切割识别顺序, 那么不同个体, 不同染色体上的 DNA 用相同酶切割时产生不同长度的 DNA 片段, 这种现象叫 RFLP. 等位片段数一般是 2 3 个, 即有酶切点和无酶切点. 4.Identification by Hybridization ( 杂交鉴定 ) Nucleic acid can be used to identify specific DNA molecules Hybridization: the process of base-pairing between complementary ssdna or RNA from two different sources.

9 Probe ( 探针 ) A labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence. The mixture being probed has typically either been separated by size on a gel, or is distributed as a library in different colonies Labeling of DNA or RNA probes radioactive labeling: display and/or magnify the signals by radioactivity Non-radioactive labeling: display and/or magnify the signals by antigen labeling antibody binding enzyme binding - substrate application (signal release)-fluorescent labeling End labeling: put the labels at the ends Uniform labeling: put the labels internally End labeling Single stranded DNA/RNA 5 -end labeling: polynucleotide kinase (PNK) 3 -end labeling: terminal transferase

10 How to label one end of a DNA: Labeling at both ends by kinase, then remove one end by restriction digestion. 5 paattc G G CTTAAp5 Uniformly labeling of DNA/RNA Nick translation labeling of DNA: DNase I to introduce random nicks into template DNA DNA pol I to remove dnmps from 3 to 5 and add new dnmp including labeled nucleotide at the 3 ends. Hexanucleotide primered labeling of DNA: Denature DNA add random hexanucleotide primers and DNA pol synthesis of new strand incorporating labeled nucleotide. Nick Translation 32P-dNTP Bio-dNTP OH p DNA dntp,dna 酶 I, DNA 聚合酶 I DNA 随机引物标记探针 32p-dNTP,Bio-Dntp 6bp primer Klenow,dNTP 变性 - 复姓 OH+ P P 3` 5` 5` 5` 3`

11 Strand-specific DNA probes: e.g. M13 DNA as template the missing strand can be resynthesized by incorporating radioactive nulceotides Strand-specific RNA probes: Hybridization probes can identify electrophoretically- separated DNAs and RNAs labeled by in vitro transcription of the desired RNA sequence. Southern blotting Electrophoresis blotting It is first proposed by Dr. Edwin Southern in Edinburgh University in 1975, and term Southern blotting is named for him. gel Hybridization membrane Major steps: electrophoresis transfer blotting molecular hybridization Southern blot analysis 43

12 DNA 样品 限制性内切酶消化 两部分工作 琼脂糖凝胶电泳 DNA 探针标记 变性 Another illustration 杂交 转移印迹胶膜 暴光 X-ray 片 45 Northern blot hybridization Can be used to identify a particular mrnas The protocol is fairly similar to that describe for southern blotting except that mrna are not needed to be digested with any enzymes An experimenter might carry out northern blot hybridization to ascertain the amount of a particular mrna present in a sample rather than its size Moreover, northern blot hybridization might be carried out to compare the relative levels of a particular transcript between tissues of an organism Northern analysis COB RNAs in S. cerevisiae bi1 bi2 bi3 bi4 bi5 Pre-mRNAs mrna 47

13 Southern and Northern blotting DNA on blot RNA on blot 1. Genomic DNA preparation RNA preparation 2. Restriction digestion - 3. Denature with alkali - 4. Agarose gel electrophoresis 5. DNA blotting/transfer and fixation RNA 6. Probe labeling 6. Hybridization (temperature) 7. Signal detection (X-ray film or antibody) Comparison of Southern, Northern and Western bolt hybridization Blot type Target Probe Applications Southern DNA DNA or RNA mapping genomic clones estimating gene numbers, etc Northern RNA DNA or RNA RNA sizes and abundance (gene expression level) Western Protein Antibodies protein size and abundance (gene expression level) Questions What are the frequencies if the recognition sequences for restriction endonucleases are four (tetrameric) and eight (octameric) nucleotides? A probe must be labeled before applied in hybridization (Why?)