《医学ppt课件大全molecular 》由会员分享，可在线阅读，更多相关《医学ppt课件大全molecular （138页珍藏版）》请在金锄头文库上搜索。
1、1,Welcome to MB Class,2,Molecular Biology of the Gene, 5/E - Watson et al. (2004),Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods,3,Ch 20: Techniques ofMolecular Biology Ch 21: Model Organisms,Part V: METHODS,4,
2、Molecular Biology Course,Chapter 20Techniques of Molecular Biology,Preparation, analysis and manipulation of nucleic acids and proteins,5,The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves. Hybridization-the base-pairing chara
3、cteristics of DNA and RNA DNA cloning- DNA polymerase, restriction endonucleases and DNA ligase PCR-Thermophilic DNA polymerase,6,Topic 1: Nucleic acids,CHAPTER20: Techniques of Molecular Biology,Separation by Electrophoresis (电泳分离) Cut by Restriction endonuclease (限制性内切酶切割) Identification by Hybrid
4、ization (杂交鉴定) Amplification by PCR (PCR扩增) DNA Cloning and gene expression (DNA克隆和基因表达) Genome sequence & analysis (基因组序列和分析),7,1. Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties.,Topic 1 Nucleic acids-separation,8,1.DNA and RNA molecules are
5、negatively charged, thus move in the gel matrix (胶支持物) toward the positive pole (正电极). 2.Linear DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules. 3.The mobility of circular DNA molecules is affected by their topological structures. The mobi
6、lity of the same molecular weight DNA molecule with different shapes is: supercoiled (超螺旋) linear (线性) nicked or relaxed (缺刻或松散),DNA gel mobility (DNA在胶上的迁移性),9,Fig 21-1: DNA is separated by gel electrophoresis,large,moderate,small,10,Gel matrix (胶支持物) is an inserted, jello-like porous material that
7、 supports and allows macromolecules to move through.,Gel matrix (胶支持物),11,Agarose (琼脂糖): a much less resolving power than polyacrylamide, but can separate DNA molecules of up to tens of kb,DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB 溴化乙锭),12,Polyacry
8、lamide (聚丙稀酰胺): has high resolving capability, and can resolve DNA that differ from each other as little as a single base pair/nucleotide. but can only separate DNA over a narrow size range (1 to a few hundred bp).,13,The electric field is applied in pulses that are oriented orthogonally (直角地) to ea
9、ch other. Separate DNA molecules according to their molecule weight, as well as to their shape and topological properties. Can effectively separate DNA molecules over 30-50 kb and up to several Mb in length.,Pulsed-field gel electrophoresis (脉冲电泳),14,Fig. 21-2 pulsed-field gel electrophoresis,Switch
10、ing between two orientations: the larger the DNA is, the longer it takes to reorient,15,RNA have a uniform negative charge as DNA does. RNA is single-stranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility. RNA can be treated with r
11、eagent such as glyoxal (乙二醛) to prevent RNA base pairing, so that its mobility correlates with the molecular weight,Electrophoresis is also used to separate RNAs,16,Why being used? -To break large DNA molecules into manageable fragments.,Nucleic acids-Restriction digestion,2. Restriction endonucleas
12、es (限制性内切酶) cleave DNA molecules at particular sites by the recognition of specific sequences,17,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. EcoRI,18,the 1st
13、such enzyme found,Escherichia coliSpecies category,R13 strain,How to name a restriction endonuclease?,EcoRI,19,The random occurrence of the hexameric (六核苷酸的) sequence: 1/4096 (4-6=1/46) What are the frequencies if the recognition sequences are four (tetrameric) and eight (octameric) nucleotides?,How
14、 to estimate the frequency of the RE in a DNA molecule or genome?,20,21,(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 natu
15、re 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.,22,sticky ends (粘性末端),blunt ends (平末端),Fig 21-4 Recognition sequences and cut sites of various endonucleases,23,3. DNA hybridization can be used to identif
16、y specific DNA molecules,Hybridization: the process of base-pairing between complementary ssDNA or RNA from two different sources.,Topic 1 Nucleic acids- DNA hybridization,24,A labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence.,Probe (探针),The mixture being probed has typically either been separated by size on a gel, or is distributed as a library in different colonies,
