【正文】 a linear DNA molecule with 6 copies of GAATTC ? it will be cut into 7 fragments which could be separated by the gel electrophoresis. (The largest fragment) (The smallest fragment) Digestionof a DNA fragment with endonuclease EcoRI 22 (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: Sau3AI, 5’GATC3’。t cut randomly. They bind to specific sequences and only cut at those sites識別回文結(jié)構(gòu) ) ? Smith, . and Wilcox, . A restriction enzyme from Hemophilus influenzae. 1. Purification and general properties. J. Mol. Biol. 51, 379 (1970). ? Kelly, ., Jr. and Smith, . A restriction enzyme from Hemophilus influenzae. 11. Base sequence of the recognition site. J. Mol. Biol. 51, 393 (1970). ? Roy, . and Smith, . The DNA methylases of Hemophilus influenzae Rd. 1. Purification and properties. J. Mol. Biol. 81, 427 (1973). ? Roy, . and Smith, . The DNA methylases of Hemophilus influenzae Rd. 11. Partial recognition site base sequences. J. Mol. Biol. 81, 445 (1973). ?Papers Daniel Nathans, 1978 Nobel Prize in Physiology or Medicine The last step in this development was taken by Dan Nathans He pioneered the application of restriction enzymes in geics and his work has been a source of inspiration for scientists who subsequently started to use restriction enzymes He constructed the first geic map using restriction enzymes by cleaving the DNA from a monkey virusSV40 ? As I look back on the last few decades of my life, I am struck by the good fortune that came my way. ? Throughout my schooling there was an abundance of opportunity and encouragement. ? Several of my teachers were remarkable individuals who had a lasting influence on me. ? At every stage of my career I have had interesting and cordial colleagues, some of whom are close friends. ? My field of research is as exciting to me as ever, and it remains essentially a cottage industry effort. ? I have had talented students who are a source of much enjoyment, and I anticipate more to e as their careers develop. ? And most important, 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. 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。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(biāo)記 )抗體抗原特異性反應(yīng) 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(nèi)切酶消化 瓊脂糖凝膠電泳 轉(zhuǎn)移印跡 膠 膜 標(biāo)記 雜交 曝光 blot 46 47 Example: 實戰(zhàn)中學(xué)習(xí)技術(shù) ? 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(biāo)記探針） 49 Southern hybridization 50 Northern hybridization ? Basic principle: the basepairing characteristics of DNA and RNA ? Application: Identify specific sequence in the p