Enzymes

1
??? ?????????
Enzymes
2
?????
??? ???????? ??? DNA?????? DNA??????
DNA?????? ????? ??? ???????
3
??????

• ?????????????DNA????DNA?????????,???????????????
  ???????? 

4
??? ????????
??????????
(Restriction endonuclease)
?????????DNA?????????????,??????DNA?????????

5
1. ??
?????????
2. ??
???
????????????????? ??5-P?3-OH??
??????
3. ??
??????????(R/M??)
6
(No Transcript)
7
(1)??(Restriction)
????????????????DNA????,??????
8
(2)??(Modification)
?????DNA???????????????,?????????????????
? Dam????(DNA Adenine Methylase)
GATC ???N6??????
? Dcm????(DNA cytosine Methylase)
CCAGG?CCTGG??????C?C5???????
9
(No Transcript)
10
(3)??????????????
? hsd R ????????
??????DNA????????,????DNA??
? hsd M ?????????
????DNA?????????????,???? DNA????
? hsd S ??????????????????
????????????????????
11
???????????
1973?H.O Smith?D. Nathans???????,??????

1. ????????????????????3???????????????,?????????

????(Escherichia coli)?Eco?? ?????(Haemophilus
influenzae)?Hin??
12
2. ??????????????,???????????????????,????????????
(??)????,????????????????????
? Hind ?d?? EcoR I ???R??
13
3. ???????????????????????,??????????????????????
?Hind ?d??????????
4. ??????,???????????????????????????R,?????M?
? R.Hind ????????? M.Hind ? ????????? ?????,R??
???
14
???????????
????????????????????????
? ???????
? ???????
? ???????
15
1. ?????????????
???M.Meselson?R.Yuan?1968?????? B?? K?????
? EcoB? EcoK?
(1)??????
????????????
EcoB TGA(N)8TGCT EcoKAAC(N)6GTGC
16
(2)????
???????????10001500 bp?????????,???????,????
Recognize site
cut
1-1.5kb
(3)????
?ATP?Mg2?SAM(S-??????)
17
2. ?????????????
???H.O. Smith?K.W. Wilcox?1970?????????????
????????Hind ? ????????
(1)??????
?????????DNA???????(?????????????????)? ?DNA?????,
?????????
18
???????(rare cutter)
????6??????????????????? ?Not I (GCGGCCGC)
??????????????,??????????????? ?Hind
????4?????? Y C?T R A ?G
GTYRAC
-3
5-
19
(2)????
?????
????DNA,??????(sticky end)?????(blunt end)??
EcoR I 5-G?AATTC-3 3-CTTAA?G-5 Pst I 5-CTGCA?G-3 3-G?ACGTC-5 ?????? EcoR V 5-GAT?ATC-3 3-CTA?TAG-5 ??????
20
1 ????(sticky ends,cohensive ends)
?????????????
?????
?) 5???(?EcoR I??)
GAATTC
5-
-3
CTTAAG
3-
-5
5-
G AATTC
-3
CTTAA G
3-
-5
21
?) 3???(?Pst I??)
CTGCAG
5-
-3
GACGTC
3-
-5
5-
-3
CTGCA G
3-
-5
G ACGTC
22
2 ???????
? ????
i)???DNA??
????????????????
???????????????
ii)???DNA?????
?????????????????????
23
(No Transcript)
24
? ????
A ????DNA????????32P???
B ???????????????????????(?dA?dT),???????????????
? ???????
???????DNA???????????
25
3 ????(blunt end)
??????????????
?EcoR V
5-
GAT?ATC
-3
3-
CTA?TAG
-5
26
4 ???????
????,?????,???????????1,?????????????
????????????????
?)??? ??DNA???? (klenow fragment)????????????
?????
27
?)??(?)? ??S1?Bal31??????????????????????,??????
??
28
5 ???(Isoschizomer)
??????,???????,??????????
? ?????
??????????? ?Hind ? ?Hsu I

• Hind ? 5-A?AGCTT-3
• 3-TTCGA?A-5
• Hsu I 5-A?AGCTT-3
• 3-TTCGA?A-5

29
? ??????
??????,?????? ?Xma I ? Sma I?
Xma I 5-C?CCGGG -3 3-GGGCC?C-5 Sma I 5-CCC?GGG-3 3-GGG?CCC-5
30
6 ???(Isocaudamers)
???????,?????????????BamH I?Bgl ??Bcl I?Xho ??
5-G?GATCC-3 3-CCTAG?G-5
BamH I
5-A?GATCT-3 3-TCTAG?A-5
Bgl ?
Bcl I
5-T?GATCA-3 3-ACTAG?T-5
5-U?GATCY-3 3-YCTAG?U-5
Xho ?
U????Y?????
31
Sau 3A
5-?GATC----3 3----CTAG?-5
????????????????????????????????
GATCT-3 A-5
5-G 3-CCTAG
BamH I
Bgl ?
5-GGATCT-3 3-CCTAGA-5
Bgl ?
BamH I
Sau 3A
32
7 ???????
????????????????????????????pH????????????????????
?
???????????????
? ??()??
??????????????????????,????()???
33
??????????!
EcoR I?BamH I??????
GAATTC
EcoR I
?????pH(gt8)???????
GAATTA?AAATTC?GAGTTC?
7
34
? ????()?????
?)????? ?)??????? ?)????? ?)?pH ?)?????,??? ?)Mn2
?Mg2?Cu2?Zn2?????????
??????,??????????????
35
8?s???????
????(shifted cleavage)
?????????????????????DNA???
Hga I
GACGCNNNNN?
5
3
CTGCGNNNNNNNNNN?
36
(3)????????????????
?????????????????????? ???????DNA??,?????? ? EcoR
I???????EcoR I????
??5-G?AATTC-3 3-CTTAA?G-5 ??5-
GAATTC-3 3-CTTAAG-5
?????????
37
(4) II??????????DNA???
???????DNA,????????????????DNA?????,?????????
?Hha?????DNA? ????DNA???50
38
3. ????????
??????????DNA,?????ATP? Mg2?SAM(S-?????)?
EcoP1 AGACC
EcoP15 CAGCAG
?????????????
39
????????????????
?? ????? ????? ?????
??????? ??????? ?????????? ?????????
???????? 3?????? ???? 2??????
???????????? ATP? Mg2?SAM Mg2 ATP? Mg2?SAM
??????????? EcoBTGA(N)8TGCT EcoKAAC(N)6GTGC ???? (? s???) EcoP1AGACC EcoP15CAGCAG
40
???? ?????????1000 bp????????? ????????????? ????????3?24-26bp?
???? ?? ? ?
DNA???? ? ?? ??
???????? ??????? ??????? ???????
????????????? ? ? ?
??????? ?? ? ?
???????? ?? ???? ????
41
??????????????
1. ?????????
????(U)????????? ???????????,?20µl??????,1h????1
µgDNA???????
???????(2-5?)????????
42
2. ????

• ?????
• ??
• ????
• ??????

43
? ????
??20µl,???????????????10???,??????????
???????
ddH2O buffer DNA ?
44

• ???II ???????????????

45
? ??
????????????
???????(????DNA),????????
????????????? ???,??????????
46
?????
????
?)???????(EDTA)???? 10mmol/L ????????(SDS)
????0.1(W/V)
?)65???20min??70???10min
?)?/????
47
? ??????
????????????? ?? ??????????
48
?????????DNA???,??????

• ???????,????????
• ??????,?????????
• ??DNA???????DNA???

?????????,??????????

• ??DNA??
• ?????
• ????????????DNA????????????????

49
(1)?????????????
3. ???????DNA?????
1986?J.A. McClarin???X????????????????????????????
??
GAATTC
CTTAAG
50
(2) ????
????DNA????????????
1
2
3
4
1
2
3
4
51
(3) ????
??????????????
1
2
3
4
1
4
??????????????????????????????????????
52
(4) ???????????
53
(No Transcript)
54
(5)???????????????
AATT ACGT AGCT ATAT CATG CCGG
?
? MaeII Hpa?cMspIc
? Alu I
?
A?T Nla ?
A?T ApoI Hind? Afl III AgeI BsrFI
A?T
A?T SspI
A?T
55
AATT ACGT AGCT ATAT CATG CCGG
A?T Nsp7524
C?G NcoI StyI Dsa I XmaI AvaI
C?G NdeI
C?G Pml I BsaAI PvuII NspBII SmaI
C?G
C?G
G?C EcoRI ApoI NgoMI BsrFI
56
AATT ACGT AGCT ATAT CATG CCGG
G?C BseHI
G?C Ecl136II EcoRV NaeI
G?C
G?C AatII SacI BanII HgiAI Bsp1286 SphI Nsp7524
T?A BspHI BspMII
T?A
T?A SnaBI BsaAI
T?A
T?A
57
CGCG CTAG GATC GCGC GGCC
? MboIc Sau3AIc
? BfaI HinpI
? BstUI DpnI HaeIII
? HhaI
A?T
A?T MluI Afl III SpeI Bgl II BstYI
A?T
A?T Eco47III StuI
A?T
58
CGCG CTAG GATC GCGC GGCC
A?T HaeII
C?G DsaI AvrII StyI EagI EaeI GdiII
C?G
C?G NspBII
C?G SacII PvuI
C?G BsiEI BsiEI
G?C BssHII NheI BamHI BstYI KasI BanI Bsp120I
59
CGCG CTAG GATC GCGC GGCC
G?C NarI BsaHI
G?C
G?C
G?C
T?A BbeI HaeII ApaI BanII Bsp1286
T?A XbaI Bcl I EaeI
T?A NruI FspI MscI
T?A
T?A
60
GTAC TATA TCGA TGCA TTAA
?
? Csp61 TaqI MseI
? RsaI
?
A?T
A?T
A?T ClaI AseI
A?T ScaI
A?T
61
GTAC TATA TCGA TGCA TTAA
A?T Nsi I
C?G Bsi WI SfcI XhoI AvaI AvaI SfcI
C?G
C?G
C?G
C?G PstI
G?C Asp718BanI Sal I ApaLI
62
GTAC TATA TCGA TGCA TTAA
G?C AccI AccI
G?C Bsrl107I HincII HpaI HincII
G?C
G?C KpnI Bsp1286 HgiAI
T?A
T?A BstBI
T?A DraI
T?A
T?A
63
? ?????????????????
4. ????????????????
1U??????1h???10µg DNA,??????2-10U/ug
DNA,??????????
? ????????1????????
? ????????50??????? ?,?-20?????
64
? ??????,??????
? ?????????,?????????
? ????????,?????
65
???????????????
1. DNA???
DNA?????????????????SDS?EDTA??????????
????

• ???DNA
• ???????,1ugDNA?10U?
• ???????
• ???????( gt20?l)

66

• ?????????(DNA)????.????RNA????????????EDTA??????
  ????????SDS????????
• DNA???????,????DNA???????????????????????????????5
  0ul??1ug DNA?

67
2. DNA??????
?????????????????
DNA???????(DNA adenine methylas ,dam)
(??GATC??A) DNA???????(DNA cytosine ethylas
,dcm)(??CCA/TGG?C)?
?????????????????????
68
3. ??
???????????????????????37 oC,????40-65
oC????DNA?15????,??12???
? ????oC ? ????oC ? ????oC
Apa I Bcl I Mae II Taq I 30 50 50 65 Apy I BstE II Mae III 30 60 55 Ban I Mae I Sma I 50 45 25
69
4. ???(Buffer)
?????????????
?????????????????
(1)????????
MgCl2?NaCl/KCl ??Mg2????? Tris-HCl
??pH,????7-7.6 ?????(DTT)
?????,?????? ??????(BSA)??????,?????
???????????
70
? ??????????????
(2)???????????DNA??
? ?????????,??3???
?)?????????????(???),??NaCl??
????,????????(???) ?)???????,???2.5????????????,
??????????????????? ?)???????????buffer,?KGB(?
??? buffer),?????????????????buffer???
71
???

• ??????????????,?????????
• ???????????????????????
• ???????????????,?????? ? ?A ????????? B
  ????????? C ????????? D ????????? E ??????????

72
??? DNA ???
??DNA???(ligase)???
???DNA??????,??????????DNA??????????
DNA???????
73
1967?,??????????????????????2?DNA????????????,?DNA
????
DNA???
?????????DNA????????????
74
??DNA ligase???
1. ??????DNA?????DNA???
(1)???????
????????,???,????
(2)T4???????
?????????????????
75
2. ????
(1)???????DNA
(2)DNA3?????-OH, 5????????(P)
(3)????
???????ATP ????? NAD
76
(4)DNA????????
Tris-HCl
50 - 100 mM,pH 7.5
MgCl2
10 mM
ATP
0.5 - 1 mM
DTT
5 mM
Volume
10 - 20 ml
T T
4 - 15 ? , 4 - 16 hr
1 U DNA???????????????15 ??? 1 ??,???? 1 mg
l-DNA(Hind ???)??????
77
?????????
1. ATP(NAD)?????AMP?
2. AMP???????????-AMP???,???????PPi (NMN) ?
3. AMP?????????-?????
ATP
PPi
H3N
AMP
OC-C-CH2-CH2-CH2-CH2-NH2
78
4. AMP???????????-?????DNA? ???5? P
?,??DNA-???????
AMP
-OH
HO-P-
AMP
-OH
O-P-
79
5. 3-OH?????????,???????,???AMP?
80
???????????????????? ?
81
?????????
1. ????
???????????37?C?
2. ????
??37??????????????
?????? 416?C
82
???????????
1. DNA?????
?????????????
?DNA???DNA??????
2. ????????????
1020?
??????????????,?????????????????
83
3. ????
????1216? ????1020?
4. ATP??
??,ATP???????0.5mmol/L.
ATP????5mmol/L,????????? ATP????7.5mmol/L,????????
???????
84
??DNA???????

• ??????DNA??
• ?????
• DNA???

85
??????DNA???????

• ??????????,????????????????????????????,??????????
  ???????,?????????????

86
???????????????
???????(10??????????)
???????????,?????????
??10 PEG8000,????????????
???????(NaCl),????150-200 mM
87
??? DNA???
DNA???(DNA polymerase)
???????????,????????????????DNA??????3-OH??,?????
?????.
88
??????????DNA???

• ????DNA???
• 2. Klenow fragment
• 3. T7 DNA???
• 4. T4 DNA???
• 5. ????T7 DNA???
• 6. ????
• 7.Taq DNA???

89
?????DNA??????
1. ????
?dNTPs????????3-OH?
2. ????
???????????????
T7 DNA????????????dNTPs??????????
????DNA?????????10??dNTPs???????????
90
3. ??DNA????????
DNA??? 3?5? ???? 5?3????? ???? ????
????DNA??? ? ? ? ?
Klenow fragment ? ? ? ?
T4 DNA??? ? ? ? ?
T7 DNA??? ? ? ? ?
????T7 DNA??? ? ? ? ?
????T7DNA??? ? ? ? ?
???? ? ? ? ?
Taq DNA??? ? ? ? ?
91
??DNA????????????
1. ????DNA??? ?
?????????????DNA??(32P??)?
(1)????DNA???????
???????
?5?3???????N?
???????????????N??5?3???????,???Klenow fragment?
92
(2)DNA???I(Pol I)?????
? ??
dNTPs(dATP?dGTP?dCTP?dTTP)
? Mg2
? ??3-OH??????
? DNA??
dNTPs
5
-OH
3
93
(3)?DNA????????
? ????(probe)
???????????????????,????????????
??
?????????
????
??????????
94
? ???????
??
?????????
5
3
??
?????????
???????????????
95
? DNA???I ????????

1. ????????

?????(nick translation )
???dNTP?,DNA??? I ????5?3??????5?3???????
96
5?3??????DNA??????DNA5?????????,??????????????D
NA?3????????????
??
??
5
3
3
5
3
5
3
5
97
5
3
???DNA??
3
5
3
5
DNase I??????
5
3
32P-dNTP
3
5
???-32P-dNTP?dNTPs
5
3
32P-dNTP
3
5
DNA Pol I??????
3
5
????????
98
2. Klenow fragment
(1)Klenow fragment???
??5?3??????3?5??????(???5?3?????)?
???????
DNA Pol I ? Klenow fragment (76KD)
99
(2)????
? 3???
?????????????3????
klenow
5
5
klenow
? DNA 3????
?3???????????dNTP?
100
???????
EcoR I
BamH I
5----G3 3----CTTAA5
5----G3 3----CCTAG5
3?????DNA??
??????????? ?-32P-dNTPs
?-32P-dATP
?-32P-dGTP
25oC 1h
5----GG3 3----CCTAG5
5----GAA3 3----CTTAA5
Klenow fragment??
5----GAATT3 3----CTTAA5
5----GGATC3 3----CCTAG5
?????DNA
101
? cDNA??????
mRNA
5
3
???
3
cDNA???
5
klenow
??
3
cDNA???
5
102
3. T4 DNA???
(1) T4 DNA??????
? ??
?T4???????????????????,??????43???
? ???
?5?3??????3?5?????(??????)?
103
? ??
A ???dNTP?,T4 DNA?????3?5?????,???3????
B ??????dNTP,?????dNTP???? C ???dNTP????,?????????
?
104
5
3
5
3
T4 DNA???
?dNTPs
5
3
5
3
T4 DNA???
?dNTPs
5
5
105
(2) T4 DNA??????
? ??????
? DNA 3????
????(???????????)
?3?5???????????????3?(???3????5???)???3????
?????5?3???????,?????????dNTP?
106
5
3
DNA????
3
5
?dNTPs
3
5
T4 DNA???(3?5??)
5
3
????32P-dNTP?dNTPs
????
3
T4 DNA???(5?3??)
5
5
3
3
5
3
5
????????????
5
3
5
3
107
a. ?????????
??
???? ????? ????????
??
????(32P??14.3?) ???? ????? ??????????
108
b. ??????
i)?????
???(biotin),?????H???R
CH2-CH-NH-CO-NH-CH-(CH2)4-COOH
???dNTP??????biotin -1,6-dUTP? biotin -1,4-dATP?
biotin -1,1-dUTP??
??????????(biotin ligase)???????????
109
?????????????DNA???
5
3
3
???DNA??
5
3
5
DNase I ??????
5
3
???-dTTP
3
5
biotin -dTTP?dNTPs
5
3
???-dTTP
DNA Pol I ??????
5
3
3
5
110
ii)?????
???dNTP??????-dUTP?,??DNA????,????????DNA???
???????????????
????????????????????? (kit)?
111
4. T7 DNA???
(1)??
?T7?????????????????,???????
? T7?????5??????
T7???5??? ?5?3????3?5??????
? ??????????
?????? ?????????????
112
(2)T7 DNA??????
? ???????
???????????????????
? 3?5??????
??????????
? ??DNA???????
??DNA????DNA???????
113
(3)T7 DNA??????
? ??????DNA??????
?M13???
? ??????
? ??????
????3????????3?????
114
5. ????T7 DNA???
(1)T7DNA????????
??3?5?????,?DNA????????????3??
(2)????T7 DNA??????
? DNA??
?????
? ??DNA 3????
? ????????
115
6. ????
??RNA?DNA???(RNA???DNA???)?
(1)??
RNA?????
????????????????????(avian myeloblastosis virus,
AMV)
(2)AMV???
???????????,???????4145?
116
??????? RNaseH???
? ??
RNaseH ?5?3?3?5???????RNA-DNA??????RNA??
RNA DNA
RNaseH
RNA DNA
117
(3)???????
? ??
RNA-DNA?????5?3DNA??????
? ??cDNA
?oligo dT???(?mRNA?polyA??????)?
mRNA 5
3
AAAAA
5
TTTTT
cDNA
118
? ??DNA??
?????(random primer)?oligo dT????
????
?????????DNA???
(???????????????)
119
??? DNA???
????????????
(terminal transferase)
?????
1. ??
2. ??
???????
3. ??
5?3DNA?????
120
? ??3OH?????????
? ?????!
? ???????DNA??3OH?????DNA?????Co2??,???????
? ?????dNTPs? ??????dNTP,????????
3
DNA 5
dTTP ,?????
TTT
121
4. ????????
(1)?????
???DNA???????????????????,??????????
5
3
5
??DNA
3
??DNA
dGTP
dCTP
?????
CCC
GGG
CCC
GGG
122
(2)??????
?????????
5
AAGCTT TTCGAA
5
Hind???
A TTCGA
AGCTT A
Klenow??
123
AAGCT TTCGA
AGCTT TCGAA
?????
dTTP
AAGCATTT TTCGA
AGCTT TTTTCGAA
AAA
??DNA
AAA
124
(3)DNA3-OH????
??
Hind???
Hind???
AAGCTTTT TTCGA
AGCTT TTTTCGAA
AAA
AAA
???????????????DNA???3?? (???-11-dUTP?)
125
??T4??????
1. ??
T4????pseT??????T4??????????????????????????????
2. ??
???????N-???,???????C-???? ???-???ATP???????DNA?RN
A?5-OH????5-OH??????
126
HO-
-OH
3
5
-OH
HO-
3
5
ATP
T4??????
3
P-
-OH
5
-P
HO-
3
5
??ATP??-????32P??,???????DNA?5??
????DNA?5????????
127
3. ?????????
DNA 5-OH???????DNA?5??
(1)????(forward reaction)
3
P-
-OH
5
3
HO-
-P
5
?????
3
HO-
-OH
5
-OH
3
HO-
5
(?-32P)ATP
??????
3
-OH
32P-
5
3
HO-
-32P
5
128
(2)???????
??????????(?-32P)ATP?ADP?,?????????(?-32P)ATP??-32
P?DNA5???????
3
P-
-OH
5
-P
3
HO-
5
??????
(?-32P)ATP?ADP
3
32P-
-OH
5
-32P
3
HO-
5
????????
129
???????
1. ???????
(1)????????
Bacterial alkaline phosphatase,BAP
???????????
??????
(2)????????
Calf intestinal alkaline phosphatase,CIP
??????????
SDS???68oC??????
130
2. ????????
????DNA(?RNA)5??????
3
P-
-OH
5
3
HO-
-P
5
?????
3
HO-
-OH
5
-OH
3
HO-
5
3. ????????
(1)??????????????
131
????????????????????
5
AAGCTT TTCGAA
Hind???
5
A-OH TTCGA-P
P-AGCTT HO-A
5
?????
132
A-OH TTCGA-OH
HO-AGCTT HO-A
OH?OH?????????,?????????
????????DNA?5??P,????????OH???
133
3
5
P-AGCTT
A-OH
??DNA
5
3
TTCGA-P
HO-A
???
-OH?-OH???
A TTCGA
AGCTT A
AGCTT
A
A
TTCGA
??????????nick,???????????
134
??? ?????( Exonucleases)
????????????????????????
????DNA???
1. ?????????I(exo I)
5?3??
??5-OH
2. ??????????(exo ?)
5?3?3?5??
??3-OH?5-P
135
????DNA???
1. ??????(exo ?)
3?5??
??3-OH
????
?DNA?????????????
-OH
5
HO-
5
exo ?
5
5
?klenow????3???
136
2. ??????(? exo)
5?3???
??5-P(?????5-OH)
????
?DNA??????(?)?
5 P-
-P 5
? exo
5
5
???????
137
3. T7??6?????
5?3???
????5-P,????5-OH?
5
5
5
5
???? exo???
?????????????
138
DNA??? ???? ????
?? ?????????I(exo I) 5?3 5-OH
?? ??????????(exo ?) 5?3 3?5 5-P 3-OH
?? ??????(exo ?) 3?5 3-OH
?? ??????( ? exo) 5?3 5-P
?? T7??6????? 5?3 5-P 5-OH
139
??? ??DNA???
??S1???
1. ??
????(Aspergillus oryzae)?
2. ??
(1)???????
(2)????
? ???Zn2 ? pH4.04.3
140
3. S1????????
(1)????RNA?DNA??? (2)????????????
??????????
S1
S1
141
??????????????!
T
TACGTA CGTACG
ATGCAT GCATGC
(3)???????????????
(4)????????DNA?RNA-DNA???
142
4. S1??????
(1)??RNA
??????(???)
DNA
????????RNA??
RNA
S1
S1
RNA
200bp
400bp
???????????????!
143
(2)?mRNA???????????
RNA??
RNA?????????200bp??400bp?
?????????????????S1???
DNA
mRNA
144
??Bal 31???
1. ??
???????(Alteromonoas espejiana)
2. ??
???????DNA(RNA)??? ???????DNA????
????DNA??????????????????
145
4. Bal31???
3. ????
Mg2?Ca2
EGTA(???????)?????Ca2,??????
????DNA????????????
Hind III
EcoR I
EcoR I
BamH I
146
Bal31?????
??Bal31????DNA,?????????EGTA????,??????
?????(??Bal31??)????????????? ??????????????,?????
???DNA?????
147
Hind III
EcoR I
EcoR I
BamH I
????????????,??????????
BamH I
Hind III
EcoR I
Marker
148
Hind III
EcoR I
EcoR I
BamH I
Bal31
Hind III
EcoR I
BamH I
EcoR I
EcoR I
BamH I
Hind III
Marker
???????????,??????????
149
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150
(No Transcript)