????(gene cloning ):?????????DNA?????DNA????????????????(?????????????-PCR)???? ????(gene expression):???????????,?????????????????????

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???? ???? ???
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????(gene cloning )?????????DNA?????DNA?????????
???????(?????????????-PCR)????????(gene
expression)???????????,?????????????????????
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????(gene engineering) ????DNA??(recombinant
DNA technique)??????????????????DNA???????????DNA
???,???????????????(?????)? ??,??????????????,??
?????,??????????????,???????.
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???????? ??DNA ????DNA???DNA??????
? ( DNA????? ????)??DNA???(??)
???
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??????????(????????,?????,
PCR??,????? ) ?
??????????????(?????,?????-Western????,????(micro
cell method)
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?l ????????
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?.????( ?) ??????? -?????? ???????????????????
?,????????????????????????DNA??,??????????????????
? ?????1.???????????(???-shotgun)????DNA??????
???????? ?????
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2.?????????????????,??????????????????,?????????,
?????????????? ??????????????? 3.?????-????????
????????????????????. mRNA????? cDNA?????
??PCR????
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Principle of PCR

• DNA polymerase use single strand DNA as template
  to synthesize new DNA in vitro,(dNTPBufferMg2,P
  rimers)
• Chain reaction Repeated cycles of reaction under
  the same condition.

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The PCR reaction
Denature 95ºC
Extend Primers 72ºC
Anneal Primers 45-68ºC
template Primers, dNTPs, Taq polymerase, Mg2
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What do we achieve by cycling temperature in the
presence primers, dNTP, Taq polymerase?
Anneal primers
Round 1
Extend primers
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The exponential amplification of the gene in PCR

• The first 4 cycles of a PCR reaction in detail.
  In the 3rd cycle two double strands of the right
  length are copied(the forward and reverse strand
  are the same in length).In the 4th cycle, 8
  double strands of the right length are copied.

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Optimum condition of PCR

• Correct sources of template DNA
• Best temperature select in three steps
• Appropriate primers in both end
• Others Mg2 Inhibitoret al.

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Primer Design Is Vital

1. Primers should usually be 18-25 bases long
2. Need as much sequence information as possible
   outside target
3. 3 end of primer is most important best to have
   G or C at end
4. Primers should have approximately equal melting
   temperatures
5. Avoid repetitive sequence( internal hairpin
   structures)
6. Aim for 40-60 GC content
7. Avoid complementarities within or between
   primers
8. Can modify 5 ends to add restriction sites etc

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TA Cloning Of PCR Products Requires A s
A
A
PCR product
Taq- yes Pfu- no
pGEM-T pCR 2.1-TOPO
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Typical Reaction Mixture25 or 50µl in a micro
Eppendorf(0.5ml) tube
COMPONENT VOLUME Final Concentration
10PCR buffer 5 µl 1
10dNTPs(2mM) 5 µl 200 µM
Forward primer(10pmols/µl ) 5 µl 1µM (50pmols/50µl)
Reverse primer(10pmols/µl ) 5 µl 1µM (50pmols/50µl)
Genomic DNA template 2 µl 1 µg
Thermostable polymerase (2U/ µl ) 0.5 µl 1 unit
H2O( to 50 µl Final volume) 27.5 µl
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The Perfect Result
Reliable PCR form every Sample
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(?)?????(Vector)??????????,????????????????,?????
????????????????(1) ?????,?????(2)??????????(3)
?????????????(4)????????????(5)??????????(6)??
??,????.
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????????? ????(plasmid vector)????????(phage
vector)?????????(cosmid vector)???????(virus
vector )??.????????(cloning vector
)?????(expression vector)?????(shuttle vector)
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1. ????
(1)?????,???????????? (2)?????????????????
????????
(3)?????????,????? (4)??????????????
???????????????,????????????????,???????pBR322
,pUC19?
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LacZ
Ampr?
o p
ori
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2. ????? ??????? (Wild type phage ,wt ?)
DNA????48.5kb (???3.2107),?????????????,??12bp???
??(cohesive end),??????????????? ???????????????
1) ?????
??????,????????????,???????????????. 2)??????
????????????. ????????????????gt11
?????EMBL3?EMBL4 ?
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3. ??????????(cosmid) ????????????????????DNA c
o s??????????.???????????????????,?????????????cos
mid????? (1)??????????????
(2)????????????? (3)???????????

(4)???,?46kb,?????40-50kb???DNA??????????????????
? ??,??????cosmid??,???????,???????????,??????????
? cosmid pHC79
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4.???? ????????????????? ???(integrated)??.?
???????????????????????,??????????????????????????
? SV40-PSV????????-?????????pLPGHL?pMSV
?? ???(transient)?????????,???????????,???????????
?????,????????????????????????????????????????????
??????
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5.????(cloning vector)
??????????????????????????????????????????????????
????????????????????DNA?????????pUC18 pUC
19. 6.????(expression vector)
????????????????????????DNA?????????,?????????????
???????
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7.????(shuttle vector)
???????(bifunctional vector)?????????????????????
?. ?????????????????????????????????????????(ARS),
??????????????????????????????????????????????,???
????????????DNA?????????,???????????,????????????
???pKSV?10?pJDB219????
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kmram2
ori
P o LacZ
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(?).??????????????????????,???DNA???????????????
???????????? 1???????????(restriction
endonuclease ) ????????DNA????????????DNA???????
2????????(1)??(2)??46?????(3)??????(palin
dromic sequence)
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????(1)?????(cohesive end)DNA?????????????DNA??
???????????,???DNA??????????????BamH? 5
-G?GATCC-3 3 -CCTAG?G-5
?
5-NNNNNN-G GATCC-NNN-3
3NNNNNN-CCTAG G-NNN-5(2)?????Sma?CCC?GGG
GGG?CCC
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4??????? (1)?????????(DNA)????.????RNA?????????
???EDTA??????????????SDS???????? DNA
???????,????DNA ????????????????????????????????50
ul??1ug DNA)(2)???????????210u/ug DNA,??????????
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(3)???????(????)????Tris-Hcl????PH???7.48.0???
???Mg2??????????2????(BME)???????(DTT)??????,??
??????NaCl ,??????NaCl????????? (4)?????????????
???????370C,??????500C?600C?650C??????????????????
?,???DNA?15????,??12???
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(?) DNA??(??)?????1?DNA??(DNA recombination)
???DNA???????????????--??????.
??DNA??????DNA???? T4DNA ligase? DNA???.
T4DN ???(T4DN ligase)????T4????E.coli???????????,
?????DNA???3?0H?5?P??????????
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(1)?????(cohesive end ligate)
(a)????????????????????????????
??????(???DNA-5?P??,?5???OH)????,??????????
(b)????????????????????????,????????????(????)?

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(2)?????(blunt end ligate)????????????????????
????,????????????,?Smal CCC?GGG ??????.
  ?????? ?????? DNA???, ??????????
PH?7.2-7.8?? ????DTT(??????,???????)ATP
14?(???16?)???
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2?????(gene transfer)???DNA??????????.???????????
??????????????????????
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(1)????????E.coli (EK??)?? ??????????????
????????????????
????????????????????????????, ??????????
?????,?????????. ???????????????,????
????????(phosphate)???.????????????????????
?????????????R-M- (?????????)?????????
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(2).??(transformation) ???DNA??????DNA???????
????????? ???????(competent
cell)?????????,?CaCl2(??)??,????????DNA?
???????DNA?????40??42????2??(??AP???????,?37???1
??)??????????????,37??????
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(3)??(trasfection) ?????????????DNA?????????
??????? ????????,????????????????????????DNA?,?
????????????????,????????????,????????????????????
??????????????????(electroportion)???????????DNA?
 
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(?)?????????1.?????????? ????????(?????)pBR32
2 ????????pUC19???(a-??).LacZ-???-??ß-????
????(C?)??pUC??-??ß-????????(N?)?a?  ????
?APr, X-gal?IPTG???
????pUC19 rDNA Z????????ß??
??(???)
?????Xgal???? pUC19DNA Z????ß??????
??(????)
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LacZ
o p
APr
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2.??????(1)??DNA????????(fingerprint)
????DNA??????????????????,???????????????????
(2) PCR????????DNA,??????????,??????????????????
?? (3)?????(nucleic acid hybridization)??????DNA?
?(probe)???????DNA???
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fig 2 .P1.fingerpringt map of recombination
plasmid 1. Molecular Standard (SPP1 DNA/
EcoR1) 2. pUC19 plasmid DNA digested with Sal I
and EcoR1 3. pUC19 plasmid DNA 4.
Recombination plasmid DNA digested with Sal I and
EcoR1 5. Recombination plasmid DNA  
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3.???????????????????-??????????????????,???????
?????????????-???????????,?????????,????????????
?,???????????,????????????.?????-????????????????
???????????? ? Western blot ????????????????SDS?
???????????NC???????????? ?????????????? 
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????????-????????????DNA????????????????????,?????
??????????????- ??????????????????????????,??????
??????????????