[自然科學(xué)]techniqueofmolecularbiology(完整版)

　　

【正文】 tant, my wife and sons have created in our home an atmosphere of joy and harmony, so essential to everything else. ? Arber discovered restriction enzymes. He postulated that these enzymes bind to DNA at specific sites containing recurring structural elements made up of specific basepair sequences. ? Smith verified Arber39。s hypothesis bacteria contain restriction enzymes with the capacity to recognize and degrade foreign DNA He purified one restriction enzyme and showed that it could cleave foreign DNA He determined the chemical structure of the regions of DNA which were cut by the restriction enzyme (The enzymes don39。s own DNA from being cut by the restriction endonucleases? ? Modification ? One of the nucleotides in bacteria DNA is modified by an methylase enzyme (甲基化酶， modification enzyme) that attaches a methyl group to one of the bases ? The restriction enzyme doesn39。s. He discovered restriction enzymes. Arber was studying an earlier known phenomenon, host controlled restriction of bacteriophages, and found that this process involved changes in the DNA of the virus. The process apparently served to form a barrier against foreign geic material. Arber showed that the phenomenon could be divided into two ponents: restriction and modification. Restriction involved a breakdown of DNA, modification was a change (= methylation) of DNA which prevented restriction. Arber postulated that both processes are catalyzed by specific restriction and modification enzymes. He proposed that DNA molecules contain specific sites with the capacity to bind both types of enzymes. These sites are created by recurring structural elements formed from specific basepair sequences. The enzymes act at these sites either by cleaving the molecule (= restriction) or by methylating it (= modification). Hamilton Smith verified Arber39。1 Molecular Biology of the Gene, 6/E Watson et al. (2022) Part I: Chemistry and Geics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 2 The revised central dogma Translation RNA processing DNA repair and rebination 基因組的保持 基因組的表達 3 Chapter 20 Techniques of Molecular Biology 4 Techniques of Molecular Biology ? separating individual macromoleculars from the myriad mixtures in cell ? dissecting the genome into manageable sized segments for manipulation and analysis of specific DNA sequences ? Techniques of molecular biology have been one of the major driving forces in the research of molecular biology 5 The methods depend upon understanding of the properties of biological macromolecules themselves. ? Hybridization(分子雜交 ） the basepairing characteristics of DNA and RNA ? DNA cloning DNA polymerase, restriction endonucleases and DNA Ligase ? PCRThermophilic DNA polymerase 6 Outline ? Topic 1: Nucleic acids Techniques ? Topic 2: Protein Techniques ? Topic 3: Techniques for studying DNAprotein interaction ? Topic 4: Techniques for studying proteinprotein interaction Basic principle Basic procedure application 7 Topic 1: Nucleic acids Techniques by Electrophoresis (電泳分離 ) by Restriction endonuclease (限制性內(nèi)切酶切割 ) by Hybridization (雜交鑒定 ) Cloning and gene expression (基因克隆和表達技術(shù)） （聚合酶鏈式反應(yīng)） 8 Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties 1. Gel Electrophoresis （ 凝膠電泳） 9 and RNA molecules are negatively charged, thus move in the gel matrix (膠支持物 ) toward the positive pole (正電極 ) DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules. DNA gel mobility (electrophoretic mobility ， DNA在膠上的遷移率 ) 10 large moderate small Run gel(跑膠 ) 11 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 (超螺旋 ) linear (線性 ) nicked or relaxed (缺刻或松散 ) 12 DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB,溴化乙錠 ) DNA is separated by gel electrophoresis 1 kb kb 2 kb 3 kb 4 kb DNA marker 13 Gel matrix (膠支持物 ) is a jello(果凍狀 ） like porous material that supports and allows macromolecules to move through. Gel matrix (膠支持物 ) Polyacrylamide (聚丙烯酰胺 ) Agarose (瓊脂糖 ) 14 Polyacrylamide (聚丙烯酰胺 ): (1)has high resolving capability(分辨力高 ), and can resolve DNA/RNA that differ from each other as little as a single base pair. (2)but can separate DNA over a narrow size range (up to a few hundred bp, 幾百 bp). 15 Agarose (瓊脂糖 ): (1)a much less resolving power than polyacrylamide (2)but can separate DNA molecules of up to tens of kb 1 kb kb 2 kb 3 kb 4 kb 16 (1) RNA have a uniform negative charge as DNA does. (2) RNA is singlestranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility. (3) RNA can be treated with reagent such as glyoxal (乙二醛 ) to prevent RNA base pairing, so that its mobility only correlates with the molecular weight Electrophoresis is also used to separate RNAs 17 2. Restriction endonucleases (限制性內(nèi)切酶 ) cleave DNA molecules at particular sites ? Why use endonucleases? ? To make large DNA molecules break into manageable fragments ? Why use restriction endonucleases? ? cleave DNA molecules at particular sites 18 ? Restriction endonucleases (RE) are the nucleases that cleave DNA at particular sites by the recognition of specific sequences.(剪刀 ) ? RE used in molecular biology typically recognize (識別 ) short (48bp) target s