[自然科學]techniqueofmolecularbiology-免費閱讀

　　

【正文】 s. Nathans uses in his experiments the small DNA from a simian virus, called SV40, but his results are of general significance. In his first munication from 1971 he showed that the restriction enzyme discovered by Smith cleaves SV40 DNA into 11 well defined fragments. In this munication Nathans also discussed other possible applications of restriction enzymes in geics and in a brilliant way predicted much of the later development. Nathan39。 NotI, 5’GCGGCCGC3’ (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 ). sticky ends (粘性末端 ) blunt ends (平末端 ) Recognition sequences and cut sites of various endonucleases 24 Cleavage of an EcoRI site: The 5’ protruding ends are said to be “sticky” because they readily anneal (退火 ) through basepairing to DNA molecules cut with the same enzyme Gene B Gene A Discovery of restriction endonucleases Research strategy: from phenomenon to mechanism Phenomenon Hypothesis Experimental proof Mechanism Practical use of restriction endonucleases Salvador E. Luria, 1969 Nobel prize in Physiology or Medicine Model: Phage infecting Phenomenon: restriction and modification in bacteria (限制與修飾現象） Werner Arber, 1978 Nobel Prize in Physiology or Medicine Discovery of restriction enzymes, which are the tools which make it possible to open the sealed book He analyzed an apparently obscure phenomenon in bacteria, called hostcontrolled modification and restriction Arber postulated that bacteria contain restriction enzymes with the capacity to recognize and degrade foreign DNA phage DNA (protect the host from foreign genes) 29 Arber’s Hypothesis ? Bacteria have the ability to restrict phage (virus) infection by cutting up the phage DNA once it39。s Nobel Prize in medicine or physiology is awarded for discoveries with far reaching consequences for geics. The task of geics is to describe and explain how genes are anized and expressed in cells and in living anisms. The discovery of restriction enzymes provided new tools for the detailed chemical analysis of the mechanism of gene action. Even though these enzymes have been available only during a few years their application to geics has already led to new and far reaching results, in particular concerning the anisation and expression of genes (= DNA) of higher animals. All work in this area carried out by many research groups all over the world, is based on the discoveries made by the three laureates. Restriction enzymes are used as tools to dissect DNA into smaller defined fragments. These can be used to determine the order of genes on chromosomes, to analyse the chemical structure of genes and to rebine genes by chemical means. Most important restriction enzymes are used to analyse the function of regions of DNA which regulate gene expression. This opens up new areas of research to study the connection between heredity and function. We can now begin to answer questions of central biological importance in developmental biology: how do genes direct the evolution of a single fertilized egg to a plete individual with many different ans? What determines that the cells within one an normally retain their specialized functions? Different diseases are expressions of disturbances in normal functions and increased knowledge in molecular geics should aid in preventing and treating malformations, hereditary diseases and cancer. Werner Arber started this field of research in Geneva during the 196039。t bind to sites where one of the bases is modified by methylation Restriction Modification 40 3. DNA hybridization can be used to identify specific DNA molecules Hybridization (雜交） : the process of basepairing between plementary ssDNA or RNA from two different sources. 41 A labeled, defined sequence used to search mixtures of nucleic acids for specific molecules containing a specific sequence which is plementary to probe Probe (探針 ) 5GAATTTGCGTATGCG3 3CTTAAACGCATACGC5 Probe 42 ? DNA hybridization using probes can be used to identify specific sequence in the plicated mixture of nucleic acids 43 Radioactive labeling: display and/or magnify the signals by radioactivity. eg. 32P Nonradioactive labeling: display and/or magnify the signals by antigen labeling – antibody binding – enzyme binding signal detection eg. DIG(地高辛標記 )抗體抗原特異性反應 Probe (探針 ) must be labelled (why labeling?) 44 Southern hybridization ? Basic principle: the basepairing characteristics of DNA and DNA ? Application: identify specific sequence in the plicated mixture of DNA, estimating gene numbers DNA 樣品 DNA 探針 變性 Xray 片 限制性內切酶消化 瓊脂糖凝膠電泳 轉移印跡 膠 膜 標記 雜交 曝光 blot 46 47 Example: 實戰中學習技術 ? Problem: identify whether the genome of one fungi contains specific gene sequence coding laccase（漆酶） ? How to use Southern hybridization to resolve this problem? 48 ? Probe: DNA sequence specific to the known laccase gene Labeled probe（標記探針） 49 Southern hybridization 50 Northern hybridization ? Basic principle: the basepairing characteristics of DNA and RNA ? Application: Identify specific sequence in the plicated mixture of RNA Detecting the expression level of specific gene mRNA contents (mRNA 豐度 ) expression level of specific gene 51 Example: 實戰中學習技術 ? Problem: measuring the expression level of laccase gene in different stages of fungal development ? How to use Northern hybridization to resolve this problem? 52 ? Probe: DNA sequence specific to the known laccase gene 53 Northern analysis The strength of hybridization signal can reflect the co