1、? 1st? 3rd? 2ndin vitroin vivoPCRRT-PCREnd modification1. Overview of Useful Enzymes for Molecular Cloning2. Restriction Endonucleases 1) Host-controlled restriction and modification; 2) Restriction enzyme and modification enzyme; 3) Types of restriction enzyme; 4) Nomenclature of restriction enzyme
2、; 5) Target site of restriction enzyme; 6) Isoschizomer; 7) Isocaudamer. 3. DNA Ligase 1) Mechanism of DNA ligase; 2) Characteristic and application of DNA ligase. 4. End Modification 1) Enzymes; 2) Double-linker; 3) Adaptors; 4) Homopolyer tailing.1. Useful Enzymes for Molecular CloningThe formatio
3、n of new combinations of heritable material by the insertion of nucleic acid molecules with some enzymes (such as Restriction Endonucleases, DNA Ligase, Polymerase), produced by whatever means outside the cell. Enzyme is the formation of bio-engineering products, usually from a wild strain or eukary
4、otes (such as yeast) to transform or optimize.Enzyme as tool in Gene manipulation In nature, there are some enzymes with specific function in many micro-organisms. Nucleic acid metabolism DNA replication and repair Degradation of non-self DNAUseful Enzyme as tool in Gene manipulationMethylaseDNA lig
5、aseDNA polymeraseRNA polymeraseKinase and phosphataseNucleaseEnzyme as tool in Gene manipulation1) Host-controlled restriction and modification2) Restriction enzyme and modification enzyme3) Types of restriction enzymes4) Nomenclature of restriction enzymes5) Target site of restriction enzymes6) Iso
6、schizomer7) Isocaudamer2. Restriction Endonuclease1) Host-controlled Restriction and ModificationPhage infects bacteriaTwo enzyme activity in host:(1) Restriction endonuclease enzyme(2) Modification methyltransferaseRestriction-Modefication (R-M) systemEOP: efficiency of platingPhage infects bacteri
7、aPlating Efficiencies of Phage Grown on E.coli Strains C, K and B When Plated on These Bacteria Strain Phage C K Blambda-C 1 10-4 10-4lambda-K 1 1 10-4lambda-B 1 10-4 1(2) The enzyme that plays the function of modification via modifying exogenetic DNA is called modification enzyme, whereas restricti
8、on enzyme exert its function no longer. The modification enzyme is methylase.2) Restriction Enzyme and Modification Enzyme3) Types of restriction enzymeRestriction and modification activitiesDual functionSingle functionDual functionRecognition and cleavage siteSeparate, SignificanceEcoR I4) Nomencla
9、ture of restriction enzymesHind Haemophilus influenzae dThe recognization sequences of type II restriction endonucleases are all palindromes. nucleotidesnucleotides.EcoRI 5-G A A T T C-3 3-C T T A A G-5 cohesive end the position of target sequence at which the restricting enzyme cuts usually protrud
10、es 5 termini or 3 terminiCohesive end EcoRI 5-G-3 5-AATTC-3 3-CTTAA-5 3-G-5 Blunt end-HaeIII 5-GG CC-3 3-CC GG-5 5) Target site of restriction enzymes Assuming a random distribution of the target sequence in the DNA sequence 4 nucleotides 44=256bp 6 nucleotides 46=4096bp （star activity；the Second ac
11、tivity) The specifity of the recognition sequence of restriction endonucleases is altered, causing extra cut. Conditions causing star activity: a.glycerin concentration; b.ionic strength; c.pH; d.organic solvent; e.bivalent cation; f.the proportion of enzyme and DNA. star activity can provide new re
12、cognition and cut site for restriction endonucleases,but commmonly cut is not complete.so it should be avioded,and the pretreatment with enzyme should be carried out under standard condition. EcoRI G AATT C_ N AATT N EcoRI* C TTAA G N TTAA NWorking condition of EcoRI： EcoRI EcoRI*Tris-HCl, 100mmol/l
13、 Tris-HCl, 25mmol/l NaCl, 50mmol/l MgCl2, 2mmol/lMgCl2, 10mmol/l pH8.5, 370CpH7.5, 370C Restriction Recognition Relaxed sequencesendonuclease sequencesBamHI G/GATCC GGATCN or GPuATCCBstI G/GATCC N/GATCNBsuI GG/CC NG/CNEcoRI G/AATTC N/AATTN or PuPuATPyPySau3A /GATC GAGC or CATCTth111I TGACN/ NACN/NNG
14、TC NNGTC GACN/NNNTC GACN/NNGNCXbaI T/CTAGA N/CTAGN or N/CTANN Rotational symmetric palindromic structureThe specifity of the recognition sequence of restriction endonucleases5353BamHIThe structure of recognition sequenceCutting sitesThe restriction endonucleases of different origin recognize the sam
15、e target site (but their cut position may be different, so the end is not always the same). 6) Isoschizomer different cut position same cut position 7) Isocaudamerenzyme, substrate, bufferin most appropriate reaction conditions, 60 min, 1g DNAActivity factor: temperature, buffer, time, reaction volu
16、me, Glycerol concentration, DNA purity and structureReaction conditions of restriction enzymes II3. DNA LigaseDNA ligase: E.coli and T4 phage can encode an enzyme, which seals single-stranded nicks between adjacent nucleotides in a duplex DNA chain. 1) Mechanism of DNA ligasenCharacteristic of DNA l
17、igaseType of DNA Ligasethe optimum temperature: 370C but at this temperature the hydrogen-bonded join between the sticky ends is unstable, so the temperature is a compromise between the enzyme action and association of the termini, the optimum temperature is 160C.2) Characteristic and application of
18、 DNA ligasePCRRT-PCREnd modification1) Enzymes2) Double Linker3) Adaptors4) Homopolymer tailing4. End Modification1) Enzymes S1 Nuclease Alkaline Phosphatase Polynucleotide KinaseSingle strand-specificEndonucleaseCohesive end into Blunt endS1 Nuclease Prevent self-cyclization Calf thymusAlkaline Pho
19、sphatase Polynucleotide Kinase（1）To prevent recircularization of vector; （2）The direction of ligation is fixed;（3）It is favoured to put the cDNA fragment under the control of promoter by using Double-Linker rather than homopolymer.2) Double Linker a preformed cohesive end, lack of P(cohesive end);a
20、preformed blunt end, with P(blunt end); （the adaptors cannt form self-polymerization）Adaptors can be ligated to the foreign DNA. The plus is then phosphorylated at the 5termini before ligated into the vector.Adaptors that are suitable for cleaving of foreign fragment have several restriction sites,
21、e.g.BamHI adaptors. Hpa II (4 base pair) 5 HO-GATCCCCGGG-OH 3 3 HO-GGGCCC-P 5 Sma l (6 base pair)3) Adaptors Terminal deoxynucleotidyltransferase add nucleotides to the 3OH termini of DNA; DNA with 3-OH of cohesive termini after pretreatment with endonuclease, is a very good substrate; dA:dT or dG:dC are used extensively to construct the tailing; the circular molecules after anneal can be directly used to infect; The gap are repaired in vivo.4) Homopolymer tailing
