第二章EnzymologyofGeneticEngineering

1、Chapter TwoEnzymology of Genetic EngineeringImportant enzyme used in genetic engineeringrestriction endonuclease (限制性核酸內(nèi)切酶限制性核酸內(nèi)切酶)DNA ligase (DNA 連接酶連接酶)DNA polymerase (DNA 聚合酶聚合酶)DNA modifying enzyme (DNA 修飾酶修飾酶)exonuclease (核酸外切酶核酸外切酶)single-strand specific endonuclease (單鏈特異核酸單鏈特異核酸內(nèi)切酶內(nèi)切酶)Lesson

2、 1 Restriction Endonuclease1. The restriction-modification systems of bacteria There are restriction-modification systems in the body of bacteria. These systems can safeguard the bacterial cell from invasion by foreign DNA using a combination of covalent modification and restriction by an endonuclea

3、se. Each species of bacteria has some kinds of restriction endonuclease which can cut double strands DNA in specific sites. Therefore, any foreign DNA (e.g. from an infecting bacteriophage or from a different species of bacteria) will be cut up and inactivated (Fig. 2.1). At the same time, bacteria

4、modifies its own DNA by methylation at specific sites (Fig. 2.1). This protects the DNA from cleavage by the corresponding restriction endonuclease. The result is that invading DNA will be cut up and inactivated, while not damaging the host DNA. . Restriction endonuclease is a kind of endo-deoxyribo

5、nuclease (內(nèi)脫氧核糖核酸酶內(nèi)脫氧核糖核酸酶), existed in the body of most strains of bacteria. They can cut double-stranded DNA at specific site and make two incisions, one through each of the sugar-phosphate backbones of t h e d o u b l e h e l i x w i t h o u t d a m a g i n g t h e nitrogenous bases. Notice three

6、 pointscan only cut double-stranded DNA, can not for single-stranded DNA can cut each strand of DNAonly cut phosphodiester bond (磷酸二酯鍵磷酸二酯鍵), without damaging the nitrogenous bases 2. The definition of restriction endonuclease Fig. 2.2 restriction endonuclase (Eco RI) cut double-stranded DNA at spec

7、ific site (GAATTC)3. The types of restriction endonuclease Restriction endonucleases are categorized into one of three general groups (Types I, II and III) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cut site relativ

8、e to the recognition sequence. Types I: They cut at a site that differs, and is some distance away, from their recognition site. Therefore, they are useless in the experiments of genetic engineering.Types II: They recognize and cut DNA at the same site and are the most commonly available and useful

9、in genetic engineering. Types III: They cut DNA about 20-30 base pairs after the recognition site. So, they are also useless in the experiments of genetic engineering. Main characters of type II restriction endonucleaseThey are composed of only one subunit. This make them only have the restriction a

10、ctivity, do not have the modification activity.Required cofactor is Mg2+The recognition site is usually undivided, palindromic and 4-8 nucleotides in length. They cut DNA in or beside the recognition site. 4. The recognition sequence (site) of type II restriction endonuclease The substrates for rest

11、riction endonuclease are more-or-less specific sequences of double-stranded DNA. This specific sequence is called recognition sequences of restriction endonuclease. Important points about the recognition sequenceMost recognition sequence are 6 base-pairs; some of them are 4, 5 or 8 base-pairs. Most

12、(not all) recognition sequences are palindromes.Recognition sequences can be unambiguous or ambiguous. Length of the recognition sequence dictates how frequently the restriction endonuclease will cut in a random sequence of DNA. Moreover, if the recognition sequence is long or rich in G/C (or A/T),

13、the corresponding restriction endonuclease will cut DNA in low frequently and was named rare cutting enzyme (稀稀切酶切酶). Different restriction endonucleases can have the same recognition sequence - such restriction endonucleases are called isoschizomers (同裂酶同裂酶). Table 2.2 Recognition sequence and cut

14、site of some kinds of REsw stands for A or T Table 2.3 different restriction endonuclease, whose recognition sequence are same, are called isoschizomers5. The cut site of type II restriction endonuclease If digesting double stranded DNA with a certain restriction endonuclease, some phosphodiester bo

15、nds can be cut up in each strand of DNA. These sites are called the cut (cleavage) site of restriction endonuclease. Important points about the cut siteMost restriction endonucleases cut double stranded DNA within its recognition sequence, some cut beside its recognition sequence. Restriction endonu

16、clease can cut double stranded DNA and generate two kinds of ends - sticky end (粘性末粘性末端端) or blunt end (平末端或鈍末端平末端或鈍末端). sticky end 5 sticky end or 3 sticky end blunt endIsocaudomers (同尾酶同尾酶) are pairs of restriction endonucleases that have slightly different recognition sequences but upon cleavage

17、generate identical sticky end. For example the enzymes Bam HI, Bgl II and Sau 3A are isocaudomers. Fig. 2.3 the cut (cleavage) sites of some kinds of restriction endonuclease in DNAFig. 2.4 the restriction endonuclease cut double stranded DNA at cut (cleavage) site Table 2.2 Recognition sequence and

18、 cut site of some kinds of REsw stands for A or T 5 sticky end (5 overhangs): The enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 5 ends. Bam HI cuts in this manner.3 sticky end (3 overhangs): Again, The enzyme asymmetrical cutting wi

19、thin the recognition site, but the result is a single-stranded overhang from the two 3 ends. Kpn I cuts in this manner.Fig. 2.5 the sticky end generated by restriction endonucleaseblunt end: Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs.

20、 SmaI is an example of an enzyme that generates blunt ends.Fig. 2.6 the blunt end generated by restriction endonucleaseFig. 2.7 the same sticky ends generated by isocaudomers6. The nomenclature of restriction endonuclease EEscherichia(genus)cocoli(species)RRY13(strain)IFirst identified(order of iden

21、tification in the bacterium) Restriction endonuclease is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain. The first letter indicated the bacterial genus, from which the restriction endonuclease is isolated. The second and third

22、letter indicated the bacterial species. The fourth letter indicated the bacterial strain. The last letter indicated the order of restriction endonuclease isolated from this strain bacterium.EcoR I7. The reaction system and influence factors of restriction endonuclease the reaction system of restrict

23、ion endonuclease Reaction system is a mixture including restriction endonuclease, double-stranded DNA and some kinds of necessary reagent. Reaction systemdouble-stranded DNA (substrate)restriction endonuclease (cut substrate in specific site)buffer (favor optimal activity of restriction endonuclease

24、)purified water (dilute the substrate and restriction endonuclease into suitable concentration)the influence factors of restriction endonuclease activity the purity of double-stranded DNA substrate The higher purity of double-stranded DNA substrate, the higher activity of restriction endonuclease. t

25、he methylation of recognition sequence Most restriction endonuclease will not cut DNA that is methylated on one or both strands of their recognition site. the reaction temperature Reaction should be carried out under optimal temperature of restriction endonuclease, usually at 37. the form of double-

26、stranded DNA The forms (shapes) of DNA: linear, circled and supercoiled. Some kinds of restriction endonucleases have different activity when they digest different forms of DNA. The cutting efficiency is maybe different when restriction endonuclease cut different recognition sequences which occupy t

27、he different positions of double-stranded DNA. the buffer of restriction endonuclease Suitable divalent cation (usually used Mg2+ as divalent cation) concentration is necessary for maintaining the optimal activity of restriction endonuclease. The buffer can maintain the restriction endonuclease at n

28、eutral reaction system (pH value is usually 7.4). Some components (-mercaptoethanol, -巰基乙醇巰基乙醇; bovine serum albumin, BSA, 牛血清白蛋白牛血清白蛋白) in buffer can increase the restriction endonuclease stability.linearcircledcircledsupercoiledActivity 20Activity 10Activity 10Activity 1Fig. 2.8 the activity of re

29、striction endonuclease is maybe different when they digest different form of DNA Recognition sequence IRecognition sequence IIRecognition sequence IIIRestriction endonucleaseaddDigest 16 hRecognition sequence IRecognition sequence IIRecognition sequence IIIcut upcut upcan not cut upFig. 2.9 the diff

30、erent cutting efficiency of restriction endonuclease G A A T T CG A A T T CG A A T T CC T T A A GC T T A A GC T T A A G8. How to digest (cut) double-stranded DNA with restriction endonuclease DNA酶切反應(yīng)酶切反應(yīng) 1. 將清潔干燥并經(jīng)滅菌的將清潔干燥并經(jīng)滅菌的eppendorf管管(最好最好0.5ml)編號(hào)，用微量移液槍編號(hào)，用微量移液槍 分別加入分別加入DNA 1g和相應(yīng)的限制性內(nèi)切酶反應(yīng)和相應(yīng)的限制

31、性內(nèi)切酶反應(yīng)10緩沖液緩沖液2l，再加，再加入重蒸水使總體積為入重蒸水使總體積為19l，將管內(nèi)溶液混勻后加入，將管內(nèi)溶液混勻后加入1l酶液，用手指酶液，用手指輕彈管壁使溶液混勻，也可用微量離心機(jī)甩一下，使溶液集中在管底。輕彈管壁使溶液混勻，也可用微量離心機(jī)甩一下，使溶液集中在管底。此步操作是整個(gè)實(shí)驗(yàn)成敗的關(guān)鍵，要防止錯(cuò)加，漏加。使用限制性內(nèi)此步操作是整個(gè)實(shí)驗(yàn)成敗的關(guān)鍵，要防止錯(cuò)加，漏加。使用限制性內(nèi)切酶時(shí)應(yīng)盡量減少其離開冰箱的時(shí)間，以免活性降低。切酶時(shí)應(yīng)盡量減少其離開冰箱的時(shí)間，以免活性降低。2. 混勻反應(yīng)體系后，將混勻反應(yīng)體系后，將eppendorf管置于適當(dāng)?shù)闹С治锷瞎苤糜谶m當(dāng)?shù)闹С治锷?

32、如插在泡沫塑如插在泡沫塑料板上料板上)，37水浴保溫水浴保溫2-3小時(shí)，使酶切反應(yīng)完全。小時(shí)，使酶切反應(yīng)完全。3. 每管加入每管加入2l 0.1mol/L EDTA(pH8.0)，混勻，以停止反應(yīng)，置于冰箱，混勻，以停止反應(yīng)，置于冰箱中保存?zhèn)溆?。中保存?zhèn)溆谩?the standard procedure example a. add double-stranded DNA (liquid) in a sterile micro-centrifuge tube, then add some volume of buffer into the same tubeb. add restriction

33、 endonuclease and purified water into the tubec. centrifugate the tube a few second, make all components move to the bottom of the tubed. incubate the tube at 37 for 1-16he. add phenol or EDTA or heat at 70 for 15 minutes, in order inactivated the restriction endonucleasef.separate DNA fragments by

34、electrophoresis and purify desired fragment Lesson 2 DNA Ligase DNA ligase was discovered in 1967. Some researchers discovered a new kind of enzyme from E.coli, which can covalently join the phosphate backbone of DNA. The natural role of DNA ligase is repairing double strand breaks (nick) in DNA mol

35、ecules. Now, DNA ligase can be obtained from some kinds of prokaryotic cell (such as E.coli), and is a very important enzyme extensively used in genetic engineering. Double-stranded DNADNA ligaseFig. 2.10 The natural role of DNA ligase is repairing double strand breaks in DNA molecules chromosomea p

36、hosphodiester bond break (nick)A ATC GCA C A A T G G TA A CGACTTGTT TCA TGDNA ligase can repairFig. 2.11 the DNA ligase product1. The definition of DNA ligase DNA ligases are vital enzymes required for important cellular processes such as DNA replication, repair of damaged DNA and recombination. The

37、 enzyme mediates the formation of phosphodiester bonds between adjacent 3-OH and 5-phosphate termini, thereby joining the nicks in double stranded DNA.Fig. 2.12 DNA ligase can join the nicks in double stranded DNA 2. The classification of DNA ligase DNA ligases can be classified into two groups depe

38、nding on their requirement for ATP or NAD+ as the cofactor. All eukaryotic and virally encoded enzymes are ATP-dependent, whereas most prokaryotic enzymes require NAD+ for their activity. The mostly used DNA ligasesT4 DNA ligase: T4 DNA ligase is derived from the T4 bacteriophage. It requires ATP as

39、 a cofactor, and can join both complementary sticky end and blunt end. E.coli DNA ligase: E.coli DNA ligase is derived from E. coli. It uses NAD+ as a cofactor, and can only join complementary sticky end. Table 2.3 The main characters of E.coli DNA ligase and T4 DNA ligaseNameDerivationCofactorJoini

40、ng functionUsefulE.coli DNA ligaseE.coliNAD+only complementary sticky endnot commonly usedT4 DNA ligaseT4 bacteriophageATPboth complementary sticky end and blunt endvery useful3. Some important points should be considered when you use DNA ligase DNA ligase can only join double-stranded DNA, can not

41、for single-stranded DNA.The nick on double-stranded DNA can be repaired by DNA ligase, while the gap can not.DNA nick(切口切口):a nick is a point in a double stranded DNA molecule where there is no phosphodiester bond between adjacent nucleotides of one strand typically through damage or enzyme action.D

42、NA gap(缺口缺口):a localized loss of one of the two strands in the double helix of DNA; a discontinuity in one of the two strands due to the loss of one or more nucleotides. Fig. 2.13 The nick on double-stranded DNA can be repaired by DNA ligase, while the gap can not.Some factors can affect the joining

43、 efficiency of DNA ligase temperature Much of early work used ligation at 10 (or ever 4 ), which needs long incubation since DNA ligase activity is low at that temperature. Fashions change, and many protocols now recommend 16 . Room temperature is often used as a compromise.the concentration of DNA

44、ligase When ligating complementary sticky end, the optimal concentration of DNA ligase is about 12 weiss unite/g DNA; for blunt end, the concentration of DNA ligase should be increased to 10100 fold. the total concentration and the ratio of DNA fragments It is important to use high concentration of

45、DNA fragments, because high DNA concentration favor the joining efficiency of DNA ligase. If we want to join two kinds of DNA fragments together, such as vector and foreign gene, the ratio of these two kinds of DNA fragments would be considered, because we want to get the product of the vector-gene

46、recombinant. If the concentration of vectors is too high, we will get a greater increase in the ligation of two vector molecules together; on the contrary, if the concentration of foreign gene is too high, then the level of foreign gene foreign gene dimers will increase. Usually, the ratio of vector

47、 to foreign gene should range from 1:3 to 3:1 (molar ratio). Fig. 2.14 the ideal vector-foreign gene recombinant molecule.the ATP concentration The ATP concentration will range from 10mol/L to 1 mmol/L. At this range, the activity of DNA ligase is stable, both complementary sticky end and blunt end

48、can be ligated. 4. How to ligate different kinds of DNA fragments different kinds of DNA fragments According to different types of ends, DNA fragments can be classified into two groups, that are sticky-ended DNA fragments and blunt-ended DNA fragments. Ligating different kinds of DNA fragments use d

49、ifferent kinds of DNA ligase and apply different kinds of methods.ligating sticky-ended DNA fragmentscomplementary sticky-ended DNA fragments Both E.coli DNA ligase and T4 DNA ligase can be used to ligate complementary sticky-ended DNA fragment. In order to decrease self-ligation of complementary st

50、icky-ended DNA fragments, before ligation, DNA fragments were treated with calf intestinal alkaline phosphatase (CIP, 小牛腸堿性磷酸酶小牛腸堿性磷酸酶) or bacteria alkalinephosphatase (BAP, 細(xì)菌堿性磷酸酶細(xì)菌堿性磷酸酶). This treatment can remove the 5-phosphates, and so make it impossible for self-ligation of complementary stic

51、ky-ended DNA fragments. After treated DNA fragments with CIP or BAP, there are two nicks in recombinant DNA molecule, which can be repaired after recombinant DNA molecule was transferred into procaryotic cell.Fig. 2.15 Both E.coli DNA ligase and T4 DNA ligase can be used to ligate complementary stic

52、ky-ended DNA fragmentsor NAD+E.coli DNA ligase orT4 DNA ligaseFig. 2.16 treated DNA fragments with CIP or BAP can decrease self-ligation recombinant moleculesNicks in the two other strands are repaired by DNA polymerase and DNA ligase in vivo:5 3 exonuclease (外外切核酸酶切核酸酶) digests away dephosphorylate

53、d nucleotide5 3 DNA polymerase (DNA聚合酶聚合酶) replaces it with a proper dNTPDNA ligase ligates the nickAlternative way for repair nick by polynucleotide kinase (多核苷酸激酶多核苷酸激酶) and DNA ligase.Fig. 2.17 DNA nicks can be repaired after recombinant DNA molecule is transferred into host cellnoncomplementary

54、sticky-ended DNA fragments If DNA is digested with different restriction endonucleases, the DNA fragments will have noncomplementary sticky end. Ligating noncomplementary sticky-ended DNA fragments can not be directly achieved by either E.coli DNA ligase or T4 DNA ligase. The strategy of ligating no

55、ncomplementary sicky-ended DNA fragments is to convert the sticky ends into blunt ends, either by filling in the complementary strand, or by trimming back the unpaired sequence. Filling in the complementary strand can be achieved by DNA polymerase, which should lack 53 exonuclease activity, such as

56、Klenow fragment of E.coli DNA polymerase I; while trimming back the unpaired sequence can be performed by other kinds of enzymes, such as exonuclease VII (外切核酸酶外切核酸酶VII, which can degrade single-stranded DNA in either the 53 or 35 direction) or S1 nuclease (S1核酸酶核酸酶, which is an endonuclease and hav

57、e cutting activity on single-stranded DNA and RNA molecules). The blunt-ended DNA fragments can be directly ligated by T4 DNA ligase, or be modified by some kinds of enzymes, then ligated by DNA ligase. Hind IIIS1 nuclease orexonuclease VIIklenow fragent ofE.coli DNA polymerase IT4 DNA ligase5AAGCTT

58、33TTCGAA55G CTTAA3 3AATTC G55A AGCTT33TTCGA A5vectorInserted DNA5A T33T A55AAGCT AGCTT33TTCGA TCGAA55TTAAG CTTAA3 3AATTC GAATT5 5G C3 3C G5S1 nuclease orexonuclease VIIklenow fragent ofE.coli DNA polymerase I5AC GT33TG CA55AAATTC GAATTT33TTTAAG CTTAAA55AAGCTC GAGCTT33TTCGAG CTCGAA55AAGCTAATTC GAATTA

59、GCTT33TTCGATTAAG CTTAATCGAA5Fig. 2.18 noncomplementary sticky ends can be converted to blunt ends and then be ligated each otherligating blunt-ended DNA fragments Blunt-ended DNA fragments can be directly ligated by T4 DNA ligase, although the efficiency is very low compared with complementary stick

60、y-ended DNA fragments. The common way to ligate blunt-ended DNA fragments is to convert blunt ends into sticky ends, this goal can be achieved through different procedures, including homopolymer tailing, adding linkers and adding adapters.homopolymer tailing ( (同聚物加尾同聚物加尾) )Terminal deoxynucleotide