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H Cloning vectors

????????????. ????? 1.?????????????? 2.???????????????? 3.??????????????? ???????? 1.???????????? 2.?????????????????????? 3.??????,???????? 4.??????????? 5.?????????? . . Cloning vectors. H1 design of plasmid vectors H2 bacteriophage vectors H3 cosmids and YACs and BACs H4

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H Cloning vectors

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    1. H Cloning vectors

    2. ???????????? ????? 1.?????????????? 2.???????????????? 3.??????????????? ???????? 1.???????????? 2.?????????????????????? 3.??????,???????? 4.??????????? 5.??????????

    3. Cloning vectors H1 design of plasmid vectors H2 bacteriophage vectors H3 cosmids and YACs and BACs H4 eukaryotic vectors

    4. Design of plasmid vectors Characteristics of plasmid vectors Alteration of plasmid vectors Type of plasmid vectors Development of plasmid vector research Familiar plasmid vector

    5. Characteristics of plasmid vectors Independent from nuclear genome Autonomously replicating nucleic acids sequence Genetic stability Eukaryotic bacterial and epiphyte Most of plasmids consist of DNA. Most of plasmids are cccDNA (covalent, closed, circle) Mw range :1 - 300 kb ??????? ??????? ?????????

    7. ???????? ??????????DNA?????????? ??DNA???????????????????? ????????????(???)??,???????????: ? ?????????? stringent plasmid 1 - 3 ?? ? ?????????? stringent plasmid 10 - 60 ??

    8. ??????? ??????????????????,????????????,?????????????,?????????????? ??????????: ? ColE1?pMB1 ??????????,??? ? pSC101?F?RP4 ??????????,??? ? p15A????????????????,??? ???????????

    9. ??????????? ????????????????,???????????????????,??????????????,?????????????????????????????? ????????????????,???????????????????,??????????????,?????????????????????????

    10. ??????? ???????????????: ????? ???????????????????????(????),?F?Col?R??? ?????? ?????????????????,?Col?R????? ??????,????????(ColE1)?????????????,????DNA??,????? bom ?mob ????

    11. ????????? ??????DNA????????????????,?????????????????????,??: ? ????:??????????????????? ? ????:???????????? ???????DNA?????????????

    12. ????? ????????????????????????????????????????,???????????,???????????????????????? ??????????????????????????????: ? pSC101:8.8 kb,???5,????????? Tcr ? ColE1:6.5 kb,???20 – 30,??????????? E1 ? RSF2124:ColE1????,??????????? Apr

    14. ????? ??????????????????????: ?????:?????????,???1000-3000,???? ?????:??pSC101,?????10,???????? ????:???????????????????? ????:?????????????????polylinker ????:???????????,????????? ????:??????????????,?????? ????:?????????????????????? ????:??????,?????????????

    15. ???? ????(1977??):????????????,?pSC101, colE1, pCR, pBR313?pBR322? ????:??????(??????),???????????????????pUC????? ????:?????????????????????,?M13mp????,?T3,T7,sp6?????,??????????????

    16. Familiar plasmid vector pBR322 Blue-white screening pUC pUC18 / 19 pKO pGEM-3Z

    17. pBR322 pBR322 has a circle dsDNA of 4.36kb, and the compositive base sequences have been found out. pBR322 has more than 100 restriction site , and scores of single restriction site for each restriction enzyme. It and its derivatives contain two antibiotic resistance genes, ampr and tetA. If a target DNA fragment is ligated into the coding region of one of the resistance genes, say tetA, the gene will become insertionally inactivated, and the presence or otherwise of a recombinant product can be determined by the antibiotic resistance exhibited by the transformants.

    22. Replica plating Inclusion of tetracycline in the original selection plate will kill off the recombinant colonies in which we are interested. The colonies grown on a normal ampicillin plate are transferred, using an abosorbent pad, to a second plate containing tetracycline. Colonies which grown on this plate probably contain vector plasmids whereas those which do not are likely to be recombinants. The latter may be picked from the ampicillin plate for further analysis.

    23. Blue-white screening A more sophisticated procedure for screening for the presence of recombinant plasmids. Only on a single transformation plate This method also involves the insertional inactivation of a gene and uses the production of a blue compound as an indicator. The gene in this case is lacZ, which encodes the enzyme ߖgalactosidase, and is under the control of the lac promoter.

    24. If the host E.coli strain is expressing the lac repressor, then expression of a lacZ gene on the vector may be induced using IPTG, and the expressed enzyme can utilize the synthetic substrate X-gal to yield a blue product. Insetional inactivation of lacZ in the production of a recombinant plasmid would prevent the development of the blue color. The transformed cells are spread on to a plate containing ampicillin, IPTG and X-gal, to yield a mixture of blue and white colonies. The white colonies have no expressed ß-galactosidase and are hence likely to contain the inserted target fragment. The blue colonies probably contain religated vector.

    26. pUC?? pUC???????pBR322???????? pUC???: ???pBR322????,?????ampr????????????pBR322?tetr??,???M13????476bp??,?LacZ ?????????????M13?????polylinker. ????

    28. ???? ?????,??2.7KB,????DNA???;????????(?????500-700???)? ??????????DNA???????LacZa,????-??????????? ??????(the multiple cloning site, MCS)????????????????? ?MCS??M13mp????????,??pUC?????????????M13mp??,??DNA???????????

    29. pUC18 / 19: ??????????? ??? 2000 - 3000 / cell ???????(MCS) ??????? lacZ’ ?????????

    31. ????? lacZ’ ?????

    33. pKO?? ??:??????(Galk)????pBR322?EcoRI-PvuII????terr? ?????? Gal + ATP ? Gal-1-P+ADP ?14C?????????,?????*Gal-1-P????? ?Galk??????????,???????????????,?14C-Gal-l-P????,?????????????? ??Galk?????????,???????pKO??,?????????,???????????????

    34. pGEM-3Z ??????????? ??? ???????(MCS) ??????? lacZ’ ??????????????????????? ??:T7?SP6???????????DNA???RNA??????,???????????????RNA???,?E.coli BL21(DE3)?

    35. ?????? ? CaCl2????? ? ??????10kb ? ???????????

    36. H2 bacteriophage vector Characteristics of bacteriophage Bacteriophage ? M13 Virus

    37. ????????????? ???????????????,???????????????,?????????,??????????????,?????????????????????? ????????????????DNA???????????????,??: ? ????????????????????? ? ???????????????????????

    38. Bacteriophage ? Bacteriophage ?, which infects E.coli cells, can be used as a cloning vector. The process of infection by phage ? Structure of phage ? ?-DNA????? ?-DNA??????? ?-DNA????????? ?-DNA??????????? ?-DNA???????

    39. ?-????????

    40. ?-???????? ??????????? ?????????-DNA?? ??dsDNA??,???????12nt(cos),?????????????? ??48502???? ?-DNA????50????

    43. ????DNA?4??? ???:A~ J?19???,??20kb,???????????????????????????????? ??? :att?int?xis,??20kb,??DNA?????,????????? ???:??10kb,??????????N?CI???????????????????????????DNA???????? O-R ????

    44. ?-DNA????? ???? ?????????? ?????? ???????????

    45. ???? ????-DNA??????51kb,?????48.5kb,????????DNA?????2.5kb?,?????????????????????????-DNA???,???????? ????-DNA???40-50%??????????????????????????,???-DNA???????: ????? ?????

    46. ?????

    47. ???? DNA ??????? DNA ?????,???????(replacement vectors)? ?????,????????????????? 9-23kb ,????????????????? ?????(?????)

    48. ?????

    50. ????????? ????????? ?????-DNA???5? EcoRI ???7?HindIII??,???????,?????1 - 2?? ?????????? ?????????DNA?????,???????????????????????,????????????????????

    51. ?????? ?????,????-DNA??????????,??????????-DNA??????????? ????-DNA?????????????????: ? ??????? Imm434 ? ??????? lacZ

    52. imm434 imm434?????????-?????????????? ??????????-?????????,??????,??????,?????; ???DNA????????,????, ?-???????????,??????

    53. lacZ lacZ????ß-?????,??????X-gal???????? ????????lacZ???,????,?????????; ?????-DNA????????

    54. ??????????? ?????(sup)???CAG(Gln)?UAG (stop)???????????????????????,???????tRNA???????????? ?????-DNA?D?E?????????????CAG??????UAG?????-DNA????????????,????????????,????????????,????????????????????,???????????????????????????????

    55. ?-DNA??????? ??????? :Charon2?Charon6??gt11 ?????:?EMBL4??gtlc??NM762?Charon40 ??????:?EMBL1??L47??1059 ?????-??????P2????????????(Spi+),??????????-DNA??red ? gam ???????????DNA?? red ? gam ,????????Spi-??,??P2?????????????????,???????

    56. ?-DNA????????? ?-DNA?????????????????????????,??????????? ??????????????????:A??(???????),E??(????),D??(????)? ????????-???????????????,??????????????????????????:?????E??,???????D???????????????????-DNA?????,????????,?????????????-DNA???,?????????????????,????????????

    58. ?-DNA??????????? ???????????????????????? ?????????l???????,37???1?? ????????,????4 -12??????????????1013 -1014 / L,??????????? ????,????? ????,???-DNA ?????????-DNA

    59. ?-DNA??????? ???????????,????????? ?????25 kb,?????????? ???-DNA????????? ???-DNA????????? ?-DNA???????????DNA??,??????????????-DNA??????????????pET???

    60. pET ?? ???? pET-5a ???? pET ??,??????????????????? T7 ???????????????????????????????????,????????????????

    63. ?????M13?????DNA M13????????? M13 ??????? M13 ??????? M13 ???????? M13 ???????? M13 ?????? M13 DNA?????

    64. M13????????? M13????????? M13 ?????????????DNA?? M13 ??????????,?????? M13 DNA??6407???? M13 DNA????10???

    65. M13 ??????? ssDNA ,6407 ?????? (GenBank ???? V00604) ???? 90% ???????????,?? 11 ?????,????????????????????????? ? ??? ? ???? ? ??? ? ??,?????????? DNA ?????? ??????? 3 ????,??????(?? ?,? ? ?),??????(???,? ? ?),????(?? ? ?????? ? ?)???? DNA ???,??? ? ??? ? ????,????? mRNA ?????

    67. M13 ??????? M13 ?????????????,? 880nm ,?? 6-7nm ?????????? 2700 ??? ? ??????????????,????? ? ????? 50 ????????? a ???????? 5 ??? ? ? 5 ??? ? ????,????????????? 5 ??? ? ??? 5 ??? ? ?????????,??????????

    68. M13 ???????? ??????,????????? DNA (??)??????????????? DNA ,?? DNA ???,?????? DNA ????? DNA (replicative form DNA) ,? RFDNA ??? ? ????, RFDNA ????,??????????? ??????????????????????,??????????????????????????????????????????

    71. M13 ?????? ?? DNA ????????????,?? M13 ?????????????????? RF DNA?RF DNA ?????????????,???????????,???????????????

    73. M13 DNA????? ????DNA?????????????????,??DNA?????????? M13????????? ?M13??????????????,?????,???????????????,??????????? M13-DNA????????????,??1.5kb?

    74. ???? ?????????????????????????????DNA? ?????????,????????????????????????,????????????: ssDNA, dsDNA, ssRNA, dsRNA RNA???????????????DNA?????,???????????????DNA???

    75. H3 cosmids and YACs and BACs The way to form cosmids Cosmid Phagemid Artificial Chromosomes

    76. ?????????????? ?-??????M13-??????????????25 kb?1.5kb,????????????????? ???????????,????????DNA???,??????????????????DNA????????????????,??????????????,??????DNA?????????????????? ??cos????????????????,????DNA????????????????

    77. ??(cosmid) ??????????-DNA cos????????????????? 1978?Collins?Hohn???? 1.8 kb??-DNA?? + pBR322?? ?????????????(colE1),????(ampr), cos ??,???????????????????? 5-7kb ??,??????? DNA ,?????31 - 45 kb??????????? cos ??,??????????????

    79. Cosmid??? ??-???,???????,???????? ??????????????? ??????,???? ????(45 kb)?????????????? ??????,???????

    80. ???(phagemid or phasmid) ???????????????????????????????????????????????????? ?????? ??M13??????,????????? ???????????????? ???????M13mp??????(10 kb) ??????DNA????????????DNA ??????,???? pUC118 / 119, pBluescript

    81. pUC118 / 119 pUC118 pUC18 + M13??? IG pUC119 pUC19 + M13??? IG

    82. pBluescript(??????) pBluescript = pUC + f1-ori + PT3 + PT7 ???????? a-??????????????????????? ??????PT3?PT7 ????????? ???????DNA? ???????RNA? ??????,??? ?????????? pBluescript?KS(ą)

    83. pBluescript?KS(ą) ??? 4 ?????????,??????????????(????????? Kpn? ? Sac? ???,? KS ? SK ??),???????????????(???,?? DNA ?????????? DNA ,? + ? - ??)? pBluescript?KS(ą) ??????? pUC18/19 ???,??? f1 ????????????? ? 

    85. ??????? ????????????????????????????? kb ?DNA?? ???????????????????????????????????,?????????????????DNA????????????,??????????,??????????,??????????????? ???????(BAC) ???????(YAC)

    86. Bacterial Artificial Chromosomes ??????????????????F?????????,????50 - 300 kb??? ?????pBACs????????????????? BAC ?????????????????,?????????????????????? pBAC????? pBACs?????: ? ???????(gene cluster)?? ? ?????????

    87. F?? ????? F ?????? 100kb ???????60??????????????????? ??F?????????DNA(1-2???/??)?????,???????????????30??????????? ??F?????,????????????????????F???F?????????????????????

    88. pBAC????? ???? DNA ?????: ????????? ????????? F ??(????)?????????? oriS ? ???? DNA ???? ATP ??????RepE ???????????????????????? ( parA , parB , ? parC )?

    90. H4 eukaryotic vectors Transfection of eukaryotic cells Shuttle vectors Yeast episomal plasmids Agrobacterium tumefaciens Ti plasmid Virus Yeast Artificial Chromosomes

    91. Transfection of eukaryotic cells DNA transfection into eukaryotic cells can be more problematic than bacterial transformation, and the efficiency to of the process is much lower. Yeast and plant cells: protoplasts Animal cells: calcium phosphate Electroporation Microinjection Gene gun

    92. ????? ??????????????????,???DNA?????????????????? ??????????????????????????????????,????????????????????????

    93. ?????????DNA????

    94. ????? ?????????????????? PDS-1000/H?????????????????? ??????????????????????????????????????

    95. ????(Shuttle vector) ?????????????????????? ?????????????????????????,??? E.coli ????????????????? ????????????

    96. ???????(YEps) ?2ľ???????? YEps???????2ľ??,??????2ľ???????? YEp13???????,??2ľ????????????LEU2, YEp13???pBR322?????,?????????????????????

    97. 2ľ?? 2ľ???????????,??6kb??,???????????70-200??,???????????????,??????????,?????????????REP1, REP2? 2ľ?????:????????????,??????????,????????????,?LEU2????ß-?????????,?????????????????LEU??????,???????????,?????LEU-2????????????????????????????????LEU-2????,????????????

    99. YEp???

    100. YEp?????????

    101. ?????Ti?? ?????????????????????,???????????????????(A. tumefaciens)? ??????Ti(tumor-inducing)?????? ??????????????????????????????????????????????,??????????Vir(Virulence region)???????,Vir??????Ti??????T-DNA????,?????????????????????T-DNA??,?????,????????? Ti??????

    102. Ti?????160~240kB?????T-DNA???15kb-30kb?Vir????36kb???????,Ti??????Con?(region encoding conjugation)?Ori?(origin of replication)?

    103. T-DNA T-DNA?????????????? ?????????????????????,?????????????????,?????? ??????????,????????:???(octopine)????(nopaline)????(agropine)????(succinamopine),???????????? LB?RB???25bp?????????,???????????????

    104. T-DNA???Vir????????????,???LB?RB??????????????????,?????T-DNA? T-DNA?LB?RB????????????,RB???????,?LB??? T-DNA??????????,????????,???????,??????????????

    105. ???????????? T-DNA??????????,??E.coli????????????Kmr? ???E.coli???????,??????????????,???Ti??????????????,????????Ti???,?????????T-DNA??????????????DNA?,Kmr???????

    106. ??????????? ??T-DNA????????????????,?????????????????????????Ti????????,????????????????????????,??????????????????????????????????,?????????Ti????Vir??,????????T-DNA????,?????????

    108. Virus Baculovirus SV40 Retroviruses

    109. ?????? ??????????? ????????????,????????????,????????? ????????????????????????????????????????????????????????????,??????? BaculoDirect™ ?????????????,???????

    110. BaculoDirect™??????????GatewayŽ??? BaculoDirect™??DNA???attR??,???????????GatewayŽ???????????? ?entry???BaculoDirect™??DNA?GatewayŽ LR Clonase™????????,??????1????????????????????????????,????????????(sf9?sf21)?

    111. SV40 SV40 infects a number of mammalian species. Its utility for transferring large fragments is limited.

    112. ????? ????????RNA???,???????,RNA????DNA,???????bp??????????,???????,?????? ??????DNA?????????????????(5’-LTR?3’-LTR),???????????????????+,?????????????gag?pol?env? ????????????DNA????10 kb,?????????????,??????????????????,????????????????????,????????????

    113. ???????????,???poi?env???gag???3’?,????????????+?????????????????,??????????????????????????????????????????????????,??????????,???????????????????????,????????????,???????????????????????????????????? ??????????????,???????????????????,???????????,?????????????

    114. Yeast Artificial Chromosomes ???????(Saccharomyces cerevisiae)??????????????,?????????????? ??????????????,?????? 90 ??;? 16 ????,???? 225-1900kb ,???14×106 bp;??? mRNA ?????? YAC??????? YAC???????350 - 400 kb ?????????? YAC ??????????

    115. YAC????????? ?????????? ????????? ??????????? ????????? ???????? ?????????

    116. ??????????

    117. pYAC4 :?? BamH? ??????,?????????????,??????????????,??????????????????????????????????????????,??????????????????????? DNA ???

    118. YAC ?????????? ?? BamH??????????????,?? EcoR?? Sma? ?????? sup4 ???????????????,?????? DNA ????????????????????,????????????????????,??????????????,???????DNA ????????? DNA ???????????? sup4 ????,????????; ??????????????????????????????????? DNA ????????????????? YAC ??? YAC ????? DNA ????????? 200-500kb,???? 1Mb ??,???? 2Mb ?

    119. ???????????? ??????????????????????????????????? ????????? ????????????????, ?:LEU?TRP?HIS?LYS?URA?ADE ??????????,?????????????? ?????? ???????????????????????

    121. ????????????? ?????????? ??????????? ????????????

    122. ?????????? ?????? ???????????(recB-,recC-,hsdR-) ??????? ????????????????(recA-) ????????? ?????????????? ??????? ??????????,??????

    123. ??????????? E.coli ??????,????????,????,????,????? ?????????????? ?????DNA??????????????????????????,?DNA???????????????

    124. ???? ??????,????????? ,????,????,????? ?????,????????? ????????????????????????????

    125. ??? ?????????,??????????,????? ?????,?????? ??????????????

    126. ??? ?????????,??????,????,????,??????????,???? ??????????????? ?????DNA?????????????????????????????????????????,?DNA??????????????????

    127. ????(??) ?????????,????????,??????,??????,???? DNA????????? ??????????????

    128. ??????(???????? CHO) ????????,??????????,???????????? ????????,???? ???????????????????????????,?DNA??????????????????

    129. ????(???????) ??????????,???????????,???????????,?????? ?????? ????????????????????????,????????

    130. ???????????? ???? ??????:C600?W3110?HB101?JM83?JM101(Aps?Tcs?Cms) ????l-DNA:LE392?ED8654 ??? ????????? ?????? CHO

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