[自然科學]techniqueofmolecularbiology(編輯修改稿)

　

【文章內容簡介】 s 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。s hypothesis. He is a biochemist and worked independently of Arber in Baltimore. In 1970 he published two classical papers which described the discovery of a restriction enzyme from the bacterium Heamophilus influenzae and characterized in detail the mechanism of its action. Other scientists before Smith had unsuccessfully tried similar experiments. The restriction enzyme from Haemophilus influenzae degrades foreign DNA to large fragments, about 1000 basepairs in size, but does not touch the DNA of the host bacterium. Most important, Smith showed that all fragments at their beginning and end had the same three basepairs showing that the enzyme had cleaved DNA wherever a specific sequence of 6 basepairs was present. This sequence was internally symmetric and was cleaved in the middle. Many other restriction enzymes have by now been characterized by others using the methodology worked out by Smith. More than 100 such enzymes are known and in most cases the same pattern is observed: a restriction enzyme recognizes certain symmetrical basepair sequences and cleaves DNA wherever these sequences occur. Different enzymes recognize different sequences and by now a battery of enzymes is available which can be used to cleave DNA at different sites in order to produce a multitude of defined fragments. Dan Nathans pioneered the application of restriction enzymes to problems of geics. He works in Baltimore at the same university as Smith. All his contributions in this area of research were made during the 197039。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。s publication from 1971 no doubt served as a major source of inspiration for scientists who subsequently started to use restriction enzymes. Two years later he described the cleavage patterns of SV40 DNA obtained with two additional restriction enzymes. He could then piece together the fragments obtained from the three cleavages and construct the plete geic map of SV40 DNA, the first obtained by a chemical method. The general approach designed by Nathans for SV40 was later used by other scientists for mapping increasingly plex DNA structures. The map of SV40 DNA was further refined by other scientists. Today we know the plete nucleotide sequence of the molecule and thus can write the plete chemical formula for all the genes of an animal virus. Nathans himself continuously contributed new ideas and developed new methods for the application of restriction enzymes to geic problems and has continuously been a main source of inspiration in this field of research. Discovery of restriction endonucleases Phenomenon Hypothesis Experimental proof Mechanism practical use of restriction endonucleases 38 How does the bacteria prevent it39。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。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