南京农业大学基因工程chapter04.pptVIP

Many restriction endonucleases recognize hexanucleotide target sites, but others cut at four, ?ve, eight, or even longer nucleotide sequences. Sau3A (from Staphylococcus aureus strain 3A) recognizes GATC, and AluI (Arthrobacter luteus) cuts at AGCT. There are also examples of restriction endonucleases with degenerate recognition sequences, meaning that they cut DNA at any one of a family of related sites. HinfI (Haemophilus in?uenzae strain Rf ), for instance, recognizes GANTC, so cuts at GAATC, GATTC, GAGTC, and GACTC. By convention, 1 unit of enzyme is de?ned as the quantity needed to cut 1 μg of DNA in 1 hour, so we need 2 units of BglII to cut 2 μg of DNA. Most restriction endonucleases, including Bgl II, work best at 37℃. Double digestion, in which the DNA is cut by two restriction endonucleases at once. partial digestion, carried out under conditions that result in cleavage of only a limited number of the restriction sites on any DNA molecule. Partial digestion is usually achieved by reducing the incubation period, so the enzyme does not have time to cut all the restriction sites, or by incubating at a low temperature (e.g., 4℃ rather than 37℃), which limits the activity of the enzyme. Figure4.16 Estimation of the sizes of DNA fragments in an agarose gel. DNA ladders Restriction endonucleases Discovery and function Type II restriction endonuclease (table 4.1) Blunt ends and sticky ends Frequency Digestion Analysing the result of RE clervage Estimation of the sizes of DNA molecules Mapping (Restriction map) Figure4.17 Using a restriction map to work out which restriction endonucleases should be used to obtain DNA fragments containing individual genes. Fig 4.18 Restriction mapping. This example shows how the positions of the Xba I, Xho I and Kpn I sites on the λ DNA molecule can be determined * Chapter 4 Manipulation of purified DNA 1??The range of DNA manipulative enzymes Enzymes for cutting DNA-restriction endonucleases Ligation-joining DNA m