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1、实验十一 的酶切,Restriction Endonucleases: An Overview,Restriction enzymes were discovered about 30 years ago during investigations into the phenomenon of host-specific restriction and modification of bacterial viruses. Bacteria initially resist infections by new viruses, and this “restriction“ of viral gr
2、owth stemmed from endonucleases within the cells that destroy foreign DNA molecules. Among the first of these “restriction enzymes“ to be purified were EcoR I and EcoR II from Escherichia coli, and Hind II and Hind III from Haemophilus influenzae. These enzymes were found to cleave DNA at specific s
3、ites, generating discrete, gene-size fragments that could be re-joined in the laboratory. Researchers were quick to recognize that restriction enzymes provided them with a remarkable new tool for investigating gene organization, function and expression. As the use of restriction enzymes spread among
4、 molecular biologists in the late 1970s, companies such as New England Biolabs began to search for more. Except for certain viruses, restriction enzymes were found only within prokaryotes. Many thousands of bacteria and archae have now been screened for their presence. Analysis of sequenced prokaryo
5、tic genomes indicates that they are common-all free-living bacteria and archaea appear to code for them. Restriction enzymes are exceedingly varied; they range in size from the diminutive Pvu II (157 amino acids) to the giant Cje I (1250 amino acids) and beyond. Among over 3,000 activities that have
6、 been purified and characterized, more than 250 different sequence-specificities have been discovered. Of these, over 30% were discovered and characterized at New England Biolabs.,The search for new specificities continues, both biochemically, by the analysis of cell-extracts, and computationally, b
7、y the analysis of sequenced genomes. Although most activities encountered today turn out to be duplicates-isoschizomers-of existing specificities, restriction enzymes with new specificities are found with regularity. Beginning in the early 1980s, New England Biolabs embarked on a program to clone an
8、d overexpress the genes for restriction enzymes. Cloning improves enzyme purity by separating enzymes from contaminating activities present in the same cells. It also improves enzyme yields and greatly simplifies purification, and it provides the genes for sequencing and analysis, and the proteins f
9、or x-ray crystallography. Restriction enzymes protect bacteria from infections by viruses, and it is generally accepted that this is their role in nature. They function as microbial immune systems. When a strain of E.coli lacking a restriction enzyme is infected with a virus, most virus particles ca
10、n initiate a successful infection. When the same strain contains a restriction enzyme, however, the probability of successful infection plummets. The presence of additional enzymes has a multiplicative effect; a cell with four or five independent restriction enzymes could be virtually impregnable.,R
11、estriction enzymes usually occur in combination with one or two modification enzymes (DNA-methyltransferases) that protect the cells own DNA from cleavage by the restriction enzyme. Modification enzymes recognize the same DNA sequence as the restriction enzyme that they accompany, but instead of cle
12、aving the sequence, they methylate one of the bases in each of the DNA strands. The methyl groups protrude into the major groove of DNA at the binding site and prevent the restriction enzyme from acting upon it. Together, a restriction enzyme and its “cognate“ modification enzyme(s) form a restricti
13、on-modification (R-M) system.In some R-M systems the restriction enzyme and the modification enzyme(s) are separate proteins that act independently of each other. In other systems, the two activities occur as separate subunits, or as separate domains, of a larger, combined, restriction-and-modificat
14、ion enzyme.,Restriction enzymes are traditionally classified into three types on the basis of subunit composition, cleavage position, sequence-specificity and cofactor-requirements. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the
15、molecular level there are many more than three different kinds. Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. Originally thought to be rare, we now know from the analysis of sequenced genomes th
16、at they are common. Type I enzymes are of considerable biochemical interest but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns.,Type II enzymes cut DNA at defined positions close to or within their recognition sequences. The
17、y produce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for DNA analysis and gene cloning. Rather then forming a single family of related proteins, type II enzymes are a collection of unrelated proteins of many different sorts. T
18、ype II enzymes frequently differ so utterly in amino acid sequence from one another, and indeed from every other known protein, that they likely arose independently in the course of evolution rather than diverging from common ancestors. The most common type II enzymes are those like Hha I, Hind III
19、and Not I that cleave DNA within their recognition sequences. Enzymes of this kind are the principle ones available commercially. Most recognize DNA sequences that are symmetric because they bind to DNA as homodimers, but a few, (e.g., BbvC I: CCTCAGC) recognize asymmetric DNA sequences because they
20、 bind as heterodimers. Some enzymes recognize continuous sequences (e.g., EcoR I: GAATTC) in which the two half-sites of the recognition sequence are adjacent, while others recognize discontinuous sequences (e.g., Bgl I: GCCNNNNNGGC) in which the half-sites are separated. Cleavage leaves a 3-hydroxyl on one side of each cut and a 5-phosphate on the other. They require only magnesium for activity and the corresponding modification enzymes require only S-adenosylmethionine. They tend to be small, with subunits in the 200350 amino acid range.,
