华东理工大学：基因工程：jygc 4

3t/ ?<?? [? 3t/ ?<?? [? yy? ?ê ?yvD fòZ ?ê ?yvD yy? 5 2 3 4 1 6 7 8 9 ×F DNA/ ?Dyy?￥'àQ / ?Dyy?￥'àQ ×F DNA/ ?Dyy?￥'e ? / ?Dyy?￥'e ? ×F DNA/ ? ?3￥'Hq / ? ?3￥'Hq ×F DNA/ ?￥?TV? / ?￥?TV? "￥y￥ X HDyó o￥y ?÷yv?? )?￥Vr ?÷yv?? )?￥Vr ?÷y? )?￥Vr ?÷y? )?￥Vr ?÷y??t%?￥Vr ?÷y??t%?￥Vr ?÷yVrát￥s ?B? ?÷yVrát￥s ?B? 4 ×F DNA/ ?￥?TV? / ?￥?TV? C ?0￥?ê?k?
_
 ?0￥?ê?k?
_
 B ×F DNAs0￥?? ?9
a9
 A DNA￥8?×F
Ma¤
 4 ×F DNA/ ?￥?TV? / ?￥?TV? M M ¤ ¤  9 _ _ A DNA￥8?×F
MD¤
 4 ×F DNA/ ?￥?TV? / ?￥?TV? ]?=M ? 3á￥T? ￥ ?¤ ]?=M ? 3á￥T? ￥ ?¤ ]á ? 3á￥T? ￥ ?¤ ]á ? 3á￥T? ￥ ?¤ ?]T? ￥ ?¤ ?]T? ￥ ?¤ T? ￥÷D T? ￥÷D |yT? ￥ ?¤ |yT? ￥ ?¤ ×F q ×F q ]?=M ? 3á￥T? ￥ ?¤ ]?=M ? 3á￥T? ￥ ?¤ 5' 5' GAATTC CTTAAG GAATTC CTTAAG EcoRI I 5' G CTTAA AATTC G 5'5' G CTTAA 5' 5' AATTC G 5' | | G CTTAA AATTC G 5' G CTTAA 5' AATTC G G CTTAA AATTC G 5' G CTTAA 5' AATTC G T4-DNA ligase li GAATTC CTTAAG 5' 5' GAATTC CTTAAG GAATTC CTTAAG 5' 5' GAATTC CTTAAG ]á ? 3á￥T? ￥ ?¤ ]á ? 3á￥T? ￥ ?¤ BamHI I 5' 5' GGATCC CCTAGG GGATCC CCTAGG 5' G CCTAG GATCC G 5' 5' T ACTAG 5' 5' GATCA T 5' | | G CCTAG GATCC G 5' T ACTAG 5' GATCA T T4-DNA ligase li TGATCC ACTAGG 5' 5' GGATCA CCTAGT 5' TGATCA ACTAGT 5' BclI G CCTAG GATCC G 5' T ACTAG 5' GATCA T TGATCC ACTAGG 5' 5' GGATCA CCTAGT ?]T? ￥ ?¤ ?]T? ￥ ?¤ BamHI I 5' 5' GGATCC CCTAGG GGATCC CCTAGG 5' G CCTAG GATCC G 5' 5' CTGCA G 5' G ACGTC 3' T4-DNA ligase li CGATCC GCTAGG 5' 5' GGATCG CCTAGC 5' CTGCAG GACGTC 5' PstI 3' T4-DNA pol Mü Mü 5' C G 5' G C Klenow l ?ü ?ü 5' GGATC CCTAG GATCC5' CTAGG CGATCC GCTAGG 5' 5' GGATCG CCTAGC |yT? ￥ ?¤ 5‘l |yT? ￥ ?¤ l 5' G CCTAG GATCC G 5' 5' G CTTAA 5' 5' 5' AATTC G T4-DNA ligase li 5' 5' Klenow l ?ü ?ü Klenow l ?ü ?ü 5' GGATC CCTAG GATCC5' CTAGG 5' GAATT CTTAA 5' 5' 5' AATTC TTAAG TdT TdTdGTP dCTP GAATTGGGGGG CTTAA AATTC GGGGGGTTAAG GGATCCCCCCC CCTAG GATCC CCCCCCCTAGG GAATTGGGGGGGATCC CTTAACCCCCCCTAGG GGATCCCCCCCAATTC CCTAGGGGGGGTTAAG 5' 5' GAATTGGGGGGGATCC CTTAACCCCCCCTAGG GGATCCCCCCCAATTC CCTAGGGGGGGTTAAG | | BamH I I BamH I I EcoR I I BamH I I |yT? ￥ ?¤ 3‘l |yT? ￥ ?¤ l CTGCA 3' G G 3' ACGTC 5' GCATG 3' C 5' C 3' GTACG T4-DNA ligase li TdT TdTdCTP dGTP GCATGCCCCCC 3' C | | Pst I Pst I C 3' CCCCCCGTACG CTGCAGGGGGG 3' G G 3' GGGGGGACGTC 5' 5' GCATGCCCCCC G C GGGGGGACGTC CTGCAGGGGGG C G CCCCCCGTACG 5' 5' GCATGCCCCCC G C GGGGGGACGTC CTGCAGGGGGG C G CCCCCCGTACG 5' 5' GCATGCCCCCCTGCAG CGTACGGGGGGACGTC CTGCAGGGGGGCATGC GACGTCCCCCCGTACG 5' 5' GCATGCCCCCCTGCAG CGTACGGGGGGACGTC CTGCAGGGGGGCATGC GACGTCCCCCCGTACG Klenow l Pst I Sph I |yT? ￥ ?¤ üh |yT? ￥ ?¤ üh C 3' G G5' G 3' C 5' C 3' G T4-DNA ligase li TdT TdTdTTP dATP GTTTTTTTTTT 3' C | | C 3' TTTTTTTTTTG CAAAAAAAAAA 3' G G 3' AAAAAAAAAAC 5' 5' GTTTTTTTTTTG CAAAAAAAAAAC CAAAAAAAAAAC GTTTTTTTTTTG S1 C3' 5' 5' GTTTTTTTTTTG CAAAAAAAAAAC CAAAAAAAAAAC GTTTTTTTTTTG F￡ F￡ GT CA T T TTTT T T TG A A AAAA A A AC CA GT A A AAAA A A AC T T TTTT T T TG TG AC CA GT T? ￥÷D T? ￥÷D BamHI I 5' 5' GGATCC CCTAGG T4-DNA ligase li Klenow l ?ü ?ü GATCC5' G5' G CCTAG 5' 5' 5' GGATC CCTAG 5' GATCC CTAGG 5' 5' GGATCGGAATTCCGATCC CCTAGCCTTAAGGCTAGG 5' 5' EcoR I I GGAATTCC CCTTAAGG EcoR I linker I li ×F q ×F q ×F q￥?l ×F q￥?l ×F q 
cμ?÷ DNA￥×Fs0 ? 
8s09 ? è? LHq/
×F qB?1 25 - 75% ×F q ^?  ?¤Q?r q￥×1·S
?ú￥×F q V[ vve? DNA×F￥a??Tb ×F q
cμ?÷ ￥×Fs0 ? 
8s09 ? è? LHq/
×F qB?1 ×F q ^?  ?¤Q?r q￥×1·S
?ú￥×F q V[ vve? ×F￥a??Tb ×F q ×F q 4ú×F q￥ZE 4ú×F q￥ZE 4ú?÷ DNA? D8￥s01
 5 : 1 - 10 : 1 4ú?÷ ? D8￥s01
 8 DNAs0 ?¤-5"? ?v
 s0 ?¤-5"? ?v
 5' 5' GAATTC CTTAAG GAATTC CTTAAG EcoRI I 5' G CTTAA p AATTC G 5' p5' G CTTAA OH 5' 5' AATTC G 5' HO | | 5' G-A-A-T-T-C C-T-T-A-A G 5' G A-A-T-T-C C-T-T-A-A-G OHHO OHHO a? ??
? a? ??
? a? ??
? a? ??
? ×F q ×F q 4ú×F q￥ZE 4ú×F q￥ZE F ] á
 F ] á
 CTGCA 3' G G 3' ACGTC 5' GCATG 3' C 5' C 3' GTACG T4-DNA ligase li TdT TdTdCTP dGTP GCATGCCCCCC 3' C | | C 3' CCCCCCGTACG CTGCAGGGGGG 3' G G 3' GGGGGGACGTC 5' 5' GCATGCCCCCC G C GGGGGGACGTC CTGCAGGGGGG C G CCCCCCGTACG 5' 5' GCATGCCCCCCTGCAG CGTACGGGGGGACGTC CTGCAGGGGGGCATGC GACGTCCCCCCGTACG Klenow l B ×F DNAs0￥?? ?9
D9
 4 ×F DNA/ ?￥?TV? / ?￥?TV? ?￥e ?D/ ? ?￥e ?D/ ? ? q ? q ?%￥ ?9 ?%￥ ?9 ?￥e ?D/ ? ?￥e ?D/ ? Ca 2+ o?￥??% )%￥? o?￥??% )%￥? Ca 2+ o?￥??%￥? a¨?ê § f|?% )
? v?? )?

 1970My ?N/ ?
e ? ^ Ca 2+ D% )?  ???/??A?2
a?ü￡ ? ? 3 l êT¨
 P%C b#
% )%N H￥ ??S? s ? o?￥??%￥? a¨?ê § f|?% )
? v?? )?

 My ?N/ ?
e ? ^ D% )?  ???/??A?2
a?ü￡ ? ? 3 l êT¨
 P%C b#
% )%N H￥ ??S ? s ? ?￥e ?D/ ? ?￥e ?D/ ? Ca 2+ o?￥??% )%￥? o?￥??% )%￥? v?? )? s ?%￥?!
 v?? )? s ?%￥?!
 100 ml )8?!à OD 600 = 0.5
 ?? l" )8 )8?!à
 ?? l" )8 ¨ 10 ml b ?￥ 10 mM CaCl 2 ·A?? )8
 ?? ·A?? )8
 ?? ¨ 1 ml b ?￥ 75 mM CaCl 2 ·A?? )8 ·A?? )8 b?b? 12 - 24l H
!¨ l H
!¨ l" )8 l" )8 ?￥e ?D/ ? ?￥e ?D/ ? Ca 2+ o?￥??% )%￥? o?￥??% )%￥? v?? )? s ?%￥é ì?
 v?? )? s ?%￥é ì?
 | 100 μl ? s ?%
F?M?? 50 ng 8￥×F | ? s ?%
F?M?? b?b??l H b?b??l H ￥? 2 sò
￡ ?
 sò
￡ ?
 y ?|?%Màb??b? 1 - 2sò y ?|?%Màb??b? sò DNA ?¤A

 ?¤A

 F? 1 ml ?7?!
? 37￥?! 1 l H
?9
 F? ?7?!
? l H
?9
 p? a￥%8?!üe é??ê p? a￥%8?!üe é??ê ?￥e ?D/ ? ?￥e ?D/ ? % )e 3é8￥? % )e 3é8￥? ê § f?% )
? j¤? )a y ? )?
¤,?÷ DNA ￥?1àp ^%C
y7? ?% )Yè?¨e 3é8
% ?Ca￥? ?
?￥ZEMé ì×F DNAs0 ê § f?% )
? j¤? )a y ? )?
¤,?÷ ￥?1àp ^%C
y7? ?% )Yè?¨ e 3é8
% ?Ca￥? ?
?￥ZEMé ì×F s0 ? )a ? )a±t%9 V¨e 3é8Eé?? ? )a ? )a±t%9 V¨e 3é8Eé?? ?￥e ?D/ ? ?￥e ?D/ ? % )e 3é8￥? % )e 3é8￥? ?]% )￥e 3é8?!ZE?DM]
[ y ? )1 è
 % )3 3éú 1?!?
? 34%￥?·A
iF??? ?[9F% )%C· ) ?￥ ù??b?!V??
 )8 ? S??? 10.3%￥?? 1·A?bDe 3é8¤(￥ ? μ ???í ￡í?ê4 ?]% )￥e 3é8?!ZE?DM]
[ y ? )1 è
 % )3 3éú 1?!?
? ￥?·A
iF??? ?[9F% )%C· ) ?￥ ù??b?!V??
 )8 ? S??? ￥?? 1·A?bDe 3é8¤(￥ ? μ ???í ￡í?ê4 % )e 3é8￥?!
 % )e 3é8￥?!
 ?￥e ?D/ ? ?￥e ?D/ ? % )e 3é8￥? % )e 3é8￥? | 0.2 - 1ml￥e 3é8??A
    ?e 3é8

F ? 10 - 20 μl DNA×F ?¤A
] HF?cμ PEG1000? Ca 2+ ￥? 1·A

 | ￥e 3é8??A
   ?e 3é8

F ? ×F ?¤A
] HF?cμ ? ￥? 1·A

 % )e 3é8￥?
 % )e 3é8￥?
 % )e 3é8￥ 3
 % )e 3é8￥ 3
 e 3é8 3￥?1"￥ ^ P% )×?é%Cb 3 + y￥%8?! é?
=c§? ??± í í e 3é8 3￥?1"￥ ^ P% )×?é%Cb 3 + y￥%8?! é?
=c§? ??± í í ?￥e ?D/ ? ?￥e ?D/ ? λ ` )8 DNA￥? ` )8 ? s ?%￥?!
 |v?? )cμ ?è
?c?$
? MgCl 2 ￥?! ??!à OD 600 = 0.5 ?
 F?ü8? z￥×F ` )8??A
￥?sò ? 3
 F?8￥?7?!
 ￥?!l H
°à?!A ? )8 3
?!Aób9F
?ape?ê ? s ?%￥?!
 |v?? )cμ ?è
?c?$
? ??!à ?
 F?ü8? z￥×F ` )8??A
￥?sò ? 3
 F?8￥?7?!
 ￥?!l H
°à?!A ? )8 3
?!Aób9F
?ape?ê ?￥e ?D/ ? ?￥e ?D/ ? è, d? è, d? |??￥é ì DNA×F ?¤AFè, d?N￥"? y?
  @Fúaè?b<vè ?￥T¨/
% )%C? %á 3?#
é ì DNA×Fs0L Vé?%= è, d?E?Te?
7O+? ?μc%C2￥ s 8% (μr
??r qμY?v ]"￥e ?
è, dE9 V¨?é ìh" L |??￥é ì ×F ?¤AFè, d?N￥"? y?
  @Fúaè?b<vè ?￥T¨/
% )%C? %á 3?#
é ì ×Fs0L Vé?%= è, d?E?Te?
7O+? ?μc%C2￥ s 8% (μr
??r qμY?v ]"￥e ?
è, dE9 V¨?é ìh" L ? q ? q ? q￥?l ? q￥?l ? q ^? ?r q￥×1·S
?l1
 ?± X8 DNAKD?Hq/é? s8%￥s0 ?b??B?￥ ? L?/
 ?? s8%K o?¤ sB?8s0
yN ? q￥?l9 V[V?1
 ?± X8 DNA?a
 s8% ¤, DNA￥? ?
' X H ?
?ê H
?¤,8×F0￥ s8%?é
 ? q ^? ?r q￥×1·S
?l1
 ?± X8 KD?Hq/é? s8%￥s0 ?b??B?￥ ? L?/
 ?? s8%K o?¤ sB?8s0
yN ? q￥?l9 V[V?1
 ?± X8 ?a
 s8% ¤, ￥? ?
' X H ?
?ê H
?¤,8×F0￥ s8%?é
 ? q ? q ? q￥?l ? q￥?l è?
 pUC18v?? )￥? q1 10 8
' ?± X pUC18 ?oμ 10 8 ?s0?é? s8%bB± X pUC18μ 3.4X10 11 ? s0
6.02X10 17 / 2686X660

9ü ^ a
 ? 3400? pUC18s 0?μB?s0é? s8% 6BZ ?
 L=?TV??
?B± X pUC183 2ml ? s ?%
v?cμ 2X10 10 ?v?? )%
9ü ^ a
 ? 200?%oμB?%?¤, pUC18 DNA è?
 v?? )￥? q1
' ?± X ?s0?é? s8%bB± X μ ? s0


9ü ^ a
 ? ? s 0?μB?s0é? s8% 6BZ ?
 L=?TV??
? X 3 ? s ?%
v?cμ ?v?? )%
9ü ^ a
 ? ?%oμB?%?¤, ? q ? q ? q￥¨o ? q￥¨o ?¨? q?×F q?? ? V[úù !9 DNA×F L? ?¨? q?×F q?? ? V[úù !9 ×F L? è?
B DNA×F L￥×F q1 20%
? q1 10 7 μg8
 è?
B
? q1 üM¤) ?a￥8? q1??￥8? 100
ठ ? üM¤) ?a￥8? q1??￥8? 
ठ ? ×F X H
3g? 8 DNAé?×F L
$ ×F X H
3g? 8 é?×F L
$ ?×F q1 100%
5?aá 3 10 4 ?×F X H31
 ?×F q1
5?aá 3 ?×F X H31
 10 4 / 10 7 X 10 -2 = 0.1 μg 8 DNA I n? L"d￥×F q1 20%
 ?[ L=g?8?1
 I n? L"d￥×F q1
 ?[ L=g?8?1
 0.1 / 20% = 0.5 μg 8 DNA 8g? ??a
?÷ DNA￥g? ' V? 10?1￥1p ? 8g? ??a
?÷ ￥g? ' V? ￥1p ? 
5 μg

 .à? V[ ?31p?  vüeé??ê 


 .à? V[ ?31p?  vüeé??ê ? q ? q ? q￥?Yy í ? q￥?Yy í 8# DNA×Fs0Z ?
 8# ×Fs0Z ?
 8' &￥?é
 8' &￥?é
 ?]￥8?]Bé s8%
? q?] ?]￥8?]Bé s8%
? q?] 8￥ bW`
 é ì￥? ?è`? qKú
ü ?M ?¤?Ta￥ é ì￥? ?è`? qKú
ü ?M ?¤?Ta￥ 8?? bW`4[?ˉ
? qB?11??!￥ 8?? bW`4[?ˉ
? qB?11??!￥ ? ?èé ì? ? ? ) ? ?èé ì? ? ? ) ??? ￥vl
 ??? ￥vl
 é ì87y
??? v
?r q? é ì87y
??? v
?r q? ? q ? q ? q￥?Yy í ? q￥?Yy í s8%Z ?
 s8%Z ?
 s8%A?D8M?￥
 è?
 pBR322?v ?? ) JM83é
? q??
?v?? ) ED8767é￥ ? q5?ú s8%A?D8M?￥
 è?
 ?v ?? ) é
? q??
?v?? ) ? q5?ú ? q ? q ? q￥?Yy í ? q￥?Yy í ?ZEZ ?
 ?ZEZ ?
 s8%￥?) ?
?YKv s8%￥?) ?
 ?YKv  s8￥1 è
  s8￥1 è
 Ca 2+ o?? 0.1 ml ? s ?% 50 ng DNA o?? ? s ?% ?ZE
 ?ZE
 Ca 2+ o?? 10 6 -10 7 / μg DNA o?? e 3é8? 10 9 ?e 3é8 50 ng DNA e 3é8? ?e 3é8 e 3é8? 10 5 -10 6 / μg DNA e 3é8? λ-DNA? 10 7 -10 8 / μg DNA ? è, d? 10 6 -10 9 / μg DNA è, d? ?%￥ ?9 ?%￥ ?9 ?9?T ?9?T ?%￥ ?9?T ^·???-a%￥ HW? !b L H
 ?9?TaaD??T} óB
?
 Ca 2+ o??a￥ 37￥?!B?l H e 3é8?a￥ 3V? λ ` )8?a￥ 30￥?!?
 ( ? ?9?T ?%￥ ?9?T ^·???-a%￥ HW? !b L H
 ?9?TaaD??T} óB
?
 o??a￥ ￥?!B?l H e 3é8?a￥ 3V? ` )8?a￥ ￥?!?
 ( ? ?9?T ?%￥ ?9 ?%￥ ?9 ?9?T￥"￥ 92?%
 P¤μ@ ? ￥?%¨??ê?? 92?%
 P¤μ@ ? ￥?%¨??ê?? ?9?Vr8s0 ￥S:y
L??ê ?9?Vr8s0 ￥S:y
L??ê Vr?÷y
L??ê?k? Vr?÷y
L??ê?k? C ?0￥?ê?k?
_
 ?0￥?ê?k?
_
 4 ×F DNA/ ?￥?TV? / ?￥?TV? 8L.S:_? 8L.S:_? X H DNA? _? X H ? _? ?÷yát_? ?÷yát_? C ?0￥?ê?k?
_
 ?0￥?ê?k?
_
 4 ×F DNA/ ?￥?TV? / ?￥?TV? ??×F q?? q? V?r? ?XK
yNA?1 ùò??ê?k?ZEus?0Dd?0a×F0Dd ×F0a"￥×F0Dd"￥×F0 ??×F q?? q? V?r? ?XK
yNA?1 ùò??ê?k?ZEus ?0 Dd?0a ×F0 Dd ×F0a "￥×F0 Dd"￥×F0 ?0 ?0 ×F0 "￥×F0 8L.S:_? 8L.S:_? F0??êE F0??êE ROP ROI Sal I BamH I Tc r Pvu I Pst I Pst I EcoR I Cla I Hind III Hind II Bal I Ap r F0??êE￥'e ?
 F0??êE￥'e ?
 pBR322 4363 bp ori F0??êE Vus?0Dd F0??êE Vus ?0 Dd ?0a×F0Dd×F0 ?0a ×F0 Dd×F0 |?÷ DNA? ? EcoRIê?
 |?÷ ? ? ê?
 d×F0? Ap r aTc r d×F0? r ×F0? Ap r aTc r ×F0? r |?÷ DNA? ? BamHIê?
 |?÷ ? ? ê?
 d×F0? Ap r aTc r d×F0? r ×F0? Ap r aTc s ×F0? 8L.S:_? 8L.S:_? F0??êE F0??êE F0??êE￥'?T
 F0??êE￥'?T
 5|?Ap?cμ Ap￥üe 5|?Ap?cμ ￥üe | Apüe ￥?0??à | üe ￥?0??à cμ Ap? Tc￥üe  cμ ? ￥üe   Apüe  3éa? Ap? Tc üe  3éa? ? üe ?é￥?0'1 üe ?é￥?0'1 Ap Ap + Tc ??Gê Gê ×F0 8L.S:_? 8L.S:_? ?!?J??êE ?!?J??êE ?!?J??êE￥'e ?
 ?!?J??êE￥'e ?
 ?!?J??êE V[us?0Dd?0
B?? ?us×F0Dd×F0be ? ^
8s0 z{? ?!Fz￥??y
7 s8%' &?????B?!F z
|?%p??cN?!Fz￥?! 
é￥ L ^?0 ?!?J??êE V[us ?0 Dd?0
B?? ?us ×F0 Dd×F0be ? ^
8s0 z{? ?!Fz￥??y
7 s8%' &?????B?!F z
|?%p??cN?!Fz￥?! 
é￥ L ^?0 8L.S:_? 8L.S:_? ?!?J??êE ?!?J??êE èn￥?!?J??êS:
 èn￥?!?J??êS:
 ??t%?i H dTTP￥??o?
 ??t%?i H ￥??o?
 ¨?v?? )￥?!S:yèê¨?? ? 3t??y trp + ¨? v?? ) ￥?!S:yèê¨?? ? 3t??y ¨?ú???t%￥?!S:yè P¨￡F}J? ¨?ú? ??t% ￥?!S:yè P¨￡F}J? ?y tk
M?￥ s8%5ê4 tk - ?J? ?y
M?￥ s8%5ê4 ?J? dCDP dTDP dTTP T R dTMP dTDP dTTP AP TK AP ?O ?O TK ￡F}J? ? ￡F}J? ? 8L.S:_? 8L.S:_? A??êE A??êE A??êE￥'e ?
 A??êE￥'e ?
 A??êE V[us?0Dd?0
9 Vus×F0 Dd×F0be ? ^
8s0 z{?A? ?y
 Vrát? P%á 3t?Q?
V7^?Oa?Gê
?v ?? )é ì pUC18z{￥ lacZ’y
??Q?
a y ? )é ì pIJ702z{￥ melCy
??Q?
? A??êE V[us ?0 Dd?0
9 Vus ×F0 Dd×F0be ? ^
8s0 z{?A? ?y
 Vrát? P%á 3t?Q?
V7^?Oa?Gê
?v ?? )é ì z{￥ y
??Q?
a y ? )é ì z{￥ y
??Q?
? 8L.S:_? 8L.S:_? A??êE A??êE A??êE￥'?T
 A??êE￥'?T
 pUC18 Ap r lacZ' ori Ap + X-gal ×F0
Ap r + lacZ -
 ×F0
r - |?÷y X H pUC18￥lacZ’S :y=?
 P- ?
N H×F 0? Ap r alacZ -
??? ) ?
7d ×F05? Ap r alacZ +
 ?? ) ? |?÷y X H S : =?
 P- ?
N H×F 0? -
??? ) ?
7d ×F05?
 ?? ) ? X H DNA? _? X H ? _? K?? ?Mm?E K?? ?Mm?E ?ì￥K?? ?Mm?Eü ^8 ??￥?÷ DNA?  é? ?Mm?s
i[ND"￥y￥X?m?1
y N ?¨??ZE???us×F0Dd×F0
7O??k? "￥×F0b???ZE¨? ?$?￥?0?ê H
y T v
 L?'9úb ?ì￥K?? ?Mm?Eü ^8 ??￥?÷ ?  é? ?Mm?s
i[ND"￥y￥X?m?1
y N ?¨??ZE???us ×F0 Dd×F0
7O??k? "￥×F0 b???ZE¨? ?$?￥?0?ê H
y T v
 L?'9úb X H DNA? _? X H ? _? K?? ?Mm?E K?? ?Mm?E ? ?3m?E
 ? ?3m?E
 pUC18 2.7 kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI I I I Ap r r lacZ’ ori B BE P 4.0 kb 1.0 kb 0.8 kb us×F0Dd×F0 us×F0Dd×F0 ¨ BamHI ?M?0é ì DNA ¨ ?M?0é ì è￡43 ?3át? è￡43 ?3át? ×F0
 2.7 kb + 4.0 kb ×F0
 + 4.0 kb d×F0
 2.7 kb d×F0
 ?T?÷ DNA? 9 ^ 2.7 kb
Kz ê¨?BM g￥ ?|é ìL??
? ?T?÷ ? 9 ^
Kz ê¨?BM g￥ ?|é ìL??
? aYVé ?k? ^?1×F0 aYVé ?k? ^?1×F0 X H DNA? _? X H ? _? K?? ?Mm?E K?? ?Mm?E ? ?3m?E
 ? ?3m?E
 pUC18 2.7 kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI I I I Ap r r lacZ’ ori S SB P 4.0 kb 1.0 kb 0.8 kb us×F0Dd×F0 us×F0Dd×F0 ?T?÷ DNA? ? pUC18￥ SphIê?)
e5 9 V¨ SphI ?3k?
??"S??ü6
 y1 SphIdè?L
? 100?ê 50 ?í
b¨ HindIII? EcoRI ó ? ?3]" V[r?"￥
?? ? ?￥Nìo# SphI￥1 / 50 ?T?÷ ? ? ê?)
e5 9 V¨ ?3k?
??"S??ü6
 y1 dè?L
? ?ê ? ó ? ?3]" V[r?"￥
?? ? ?￥Nìo# X H DNA? _? X H ? _? K?? ?Mm?E K?? ?Mm?E ? ?3m?E
 ? ?3m?E
 pUC18 2.7 kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI I I I Ap r r lacZ’ ori B BE P 4.0 kb 1.0 kb 0.8 kb us"￥×F0Dd"￥×F0 us"￥×F0Dd"￥×F0 ¨ EcoRI ?M?0é ì DNA ¨ ?M?0é ì "￥×F0
 3.0 kb + 3.7 kb "￥×F0
 3.0 kb + 3.7 kb ?
 1.0 kb + 5.7 kb ?
 1.0 kb + 5.7 kb ¨ PstI ?M?0é ì DNA ¨ ?M?0é ì "￥×F0
 3.2 kb + 3.5 kb "￥×F0
 3.2 kb + 3.5 kb ?
 0.8 kb + 5.9 kb ?
 0.8 kb + 5.9 kb X H DNA? _? X H ? _? K?? ?Mm?E K?? ?Mm?E ? ?3m?E
 ? ?3m?E
 pUC18 2.7 kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI I I I Ap r r lacZ’ ori B BE 4.0 kb 2.0 kb 2.0 kb us"￥×F0Dd"￥×F0 us"￥×F0Dd"￥×F0 m U￥ X H?
?0é ì DNA¨ EcoRI ?M 7a
oC 2.0 kb? 2.7 kb H{0
 L=  2.0 kb￥H{?cμ ??]￥ s0 m U￥ X H?
?0é ì ¨ ?M 7a
oC ? H{0
 L=  ￥H{?cμ ??]￥ s0 2.7 kb 2.0 kb E X H DNA? _? X H ? _? K?? ?Mm?E K?? ?Mm?E ?s ?3m?E
 ?s ?3m?E
 2.2 kb PstI EcoRI BamHI I BBE 4.4 kb 1.2 1.8 kb EB 0.6 0.8 ?×Fé ìü ?3a
sY¤?/ +F ? 
 ?×Fé ìü ?3a
sY¤?/ +F ? 
 BamHI? ?3
 0.6 0.8 1.8 3.4 kb ? ?3
 0.6 0.8 1.8 3.4 kb BamHI+EcoRI? ?3
 0.6 0.8 1.8 1.2 2.2 kb ? ?3
 0.6 0.8 1.8 1.2 2.2 kb ?
 1.2 kb? ￥iV ü?? ê?B ?
 ? ￥iV ü?? ê?B BamHI?s ?3
 1.4 2.6 4.0 kb ?s ?3
 1.4 2.6 4.0 kb ?
 1.4 kb￥? A1 0.6 kb? 0.8 kb ?
V ?
 ￥? A1 ? ?
V ü ? ^ ?B￥
] ?
 2.6 kb￥? A1 0.8 ü ? ^ ?B￥
] ?
 ￥? A1 kb? 1.8 kb ?
V ü ? ^ ?B￥
Ka
 ? ?
V ü ? ^ ?B￥
Ka
 4.0 kb￥? A1 0.6 kb? 3.4 kb ?
V ü ? ￥? A1 ? ?
V ü ? ^ ?B￥ ^ ?B￥ X H DNA? _? X H ? _? ) ? ` )êeê?E ) ? ` )êeê?E ) ? ` )êeê?E￥yTe ? ^?  X H￥"￥ y DNA? ￥]÷?  !9i???
[N ?ù?êcμ "￥y￥"￥×F0b?
 DNA]÷? -W￥+s?o ?? ^?/ ?￥' ? ?G  ) ? ` )êeê?E￥yTe ? ^?  X H￥"￥ y ? ￥ ]÷?  !9i?? ?
[N ?ù?êcμ "￥y￥ "￥×F0 b?
 ]÷? -W￥+s?o ?? ^?/ ?￥' ? ?G  X H DNA? _? X H ? _? ) ? ` )êeê?E ) ? ` )êeê?E ) ?eê?E￥'?T
 ) ?eê?E￥'?T
 ?? ?? ¨b ?8? í ?? X Hüe ¨b ?8? í ?? X Hüe !′ !′ ? ? ?; ?; ¨ 0.4N￥NaOH·A????  ¨ ·A????  ¨ SSC·A??b!??  ¨ ·A??b!??  80￥??%???  ￥??%???   ??cμ?￥?·A??  ??cμ?￥?·A?? ¨ SSC-SDSA!′?  ¨ A!′?  ¨ X;??-? ?; ¨ ;??-? ?; X H DNA? _? X H ? _? ) ? ` )êeê?E ) ? ` )êeê?E ??￥?!
 ¨? ) ? ` )êeê?￥?A? ?@/ Hq
 ¨? ) ? ` )êeê?￥?A? ?@/ Hq
 ? y2
? y DNA V¨aM?
 ? y2
? y V¨aM?
 @é
à  12?a
 @é
à  =??co?u =??co?u ?￥?!ZE ?÷ ?
 ?￥?!ZE ?÷ ?
 |y?? |y?? cDNA?? ?? ]÷?  ]÷?  mRNA GACCTAAAGCGGATCGTAGGTC GACCTA CTGGAT A A G C T G G G C A X H DNA? _? X H ? _? ) ? ` )êeê?E ) ? ` )êeê?E ??￥S:
 T4-PNPo?￥ S: ??￥S:
 o?￥ S: 5‘ HO 3‘ HO OH 3‘ OH 5‘ T4-PNP Mg 2+ pppATP
γ- 32 P-ATP
 pppA 5‘ p 3‘ HO OH 3‘ p 5‘ X H DNA? _? X H ? _? ) ? ` )êeê?E ) ? ` )êeê?E ??￥S:
I c ?o?￥Q cS: ??￥S:
 I c ?o?￥Q cS: 3’AAAAAAAAAAACCAGCTTCC···GAACTGATTTAGGCT 5’mRNA Q c ? Q c ? Mg 2+ dNTP + pppdATP
α- 32 P-dATP
 dNTP + pppdATP
5’TTTTTTTTTTT 5’mRNA 3’cDNA 3’AAAAAAAAAAACCAGCTTCC···GAACTGATTTAGGCT 5’TTTTTTTTTTTGGTCGAAGG···CTTGACTAAATCCGA TTTTTTTTTTTGGTCGAAGG CTTGACTAAATCCGA X H DNA? _? X H ? _? ) ? ` )êeê?E ) ? ` )êeê?E ??￥S:
 DNA ? ?o?￥? Y-MS: ??￥S:
 ? ?o?￥? Y-MS: 5‘ … G-C-T-C-A-G-C-T-G-G-A-G-T… 3’ -C-T-C-A-G-C-T-G-G-A-G-T… 3’ 3‘ … C-G-A-G-T-C-G-A-C-C-T-C-A… 5‘ -G-A-G-T-C-G-A-C-C-T-C-A… 5‘ Mg 2+ 5‘ dNTP 5‘ ppp dA
α- 32 P-dATP
 ppp 5‘ … G-C-T-C A-G-C-T-G-G-A-G-T… 3’ -C-T-C A-G-C-T-G-G-A-G-T… 3‘ … C-G-A-G-T-C-G-A-C-C-T-C-A… 5‘ -G-A-G-T-C-G-A-C-C-T-C-A… 5‘ 5‘ … G-C-T-C-A-G-C-T-G-G-A-G-T… 3’ -C-T-C-A-G-C-T-G-G-A-G-T… 3’ 3‘ … C-G-A-G-T-C-G-A-C-C-T-C-A… 5‘ -G-A-G-T-C-G-A-C-C-T-C-A… 5‘ DNase I DNA pol I X H DNA? _? X H ? _? ) ? ` )êeê?E ) ? ` )êeê?E ??￥S:
 ABC?;S: ??￥S:
 ?;S: GCTTGAGCAGTAACCTG ?;? ?;? Biotin 3t í 3t í Avidin 3t í2??a 3t í2??a ?Q ?¤D ?Q ?¤D X H DNA? _? X H ? _? ) ? ` )êeê?E ) ? ` )êeê?E ??￥S:
 ABCA? ?S: ??￥S:
 A? ?S: GCTTGAGCAGTAACCTG A? ? A? ? Biotin 3t í 3t í Avidin 3t í2??a 3t í2??a ?Q ?¤D ?Q ?¤D 3??t 3??t t?át t?át X H DNA? _? X H ? _? ) ? ` )êeê?E ) ? ` )êeê?E ??￥S:
1ú?"dS: ??￥S:
 1ú?"dS: dUTP- ?¤D -?? Fe ?¤D ?? Fe digoxigenin DIG F8 -A? ?? óˉ?t F8 A? ? ? óˉ?t X H DNA? _? X H ? _? DNA? sE ? sE ?? ????E￥'e ?
 ?? ????E￥'e ?
 DNA? ?? ^ú?k?"￥y￥×1 m
"-S=  @? ?¨ Sanger? ü￥ ?? ??E?? DNA? 
yTe ? ^ DNA ?Q?￥é??
YVê?+s????? DNA?  1o/ ?μ
 ? ?? ^ú?k?"￥y￥×1 m
"-S=  @? ?¨ ? ü￥ ?? ??E ?? ? 
yTe ? ^ ?Q ￥é??
YVê?+s????? ?  1o/ ?μ
 DNA y ?Q? H￥?? ??
ddNTP
 y ?Q? H￥?? ??
d??`?è￡￥úsO q
600 bp / 601 bp
 d??`?è￡￥úsO q
è71?_?a: caI ?? è71?_?a: caI ?? ¨? DNA??￥B? k4?
Kit
 ??￥B? k4?
X H DNA? _? X H ? _? DNA? sE ? sE ?? ????E￥'?T
 ?? ????E￥'?T
 A C G T ssDNA ssDNA ssDNA ssDNA Primer Primer Primer Primer Klenow Klenow Klenow Klenow dNTPs dNTPs dNTPs dNTPs ddATP ddCTP ddGTP ddTTP A G TC DNA ?Q? ?Q? d??`?è￡ d??`?è￡ X H DNA? _? X H ? _? DNA? sE ? sE ?? ????E￥'Q?
 ?? ????E￥'Q?
 Klenow OH 3'5' P T dNTP + ddATP TTTTT OH 3'5' P A G TC GGCATTGCGTTACTAGTCCAGTACA ?÷yát_? ?÷yát_? ?aé 3t????E ?aé 3t????E ?x ?"￥y￥k?
 ?x ?"￥y￥k?
 ?x ??|?x ￡31?b|4· ?x ??|?x ￡31?b|4· ?x ??|?x ￡31?b|4·? ￡ ?x ??|?x ￡31?b|4·? ￡ ￥?xF?%8?!=
?!?
$b| ￥?xF?%8?!=
?!?
$b| ?k?￥×F X Hp?N?! 
c"￥y￥"￥×F0 V ?k?￥×F X Hp?N?! 
c"￥y￥ "￥×F0 V Vr￥?x ?s ??
i (  ??
] H?3?x1 V·? Vr￥?x ?s ??
i (  ??
] H?3?x1 V·? ￥?byN
"￥×F0 ) ??????i ü?b?Ti ￥?byN
 "￥×F0 ) ??????i ü?b?Ti ü?? üA
? Va?!e ¨?
 P?x ?u×? ??'?
 ü?? üA
? Va?!e ¨?
 P?x ?u×? ??'?
 ^?Oab ^?Oab ?a ?a?[ ??"￥y9 V?¨ ? ?￥ZEé?k? ?a ?a?[ ??"￥y9 V?¨ ? ?￥ZEé?k? ?÷yát_? ?÷yát_? ?aé 3t????E ?aé 3t????E F ) í F?y￥k?
 F ) í F?y￥k?
 F ) í F?yVr￥?aé? P s8%á 3? F 3 í ￥ F?b N
o3a?!e?F? a  F 3 í
 ? ? é￥ ) ?' ^cμ"￥y￥"￥×F0  L=?TV??
?? F 3 íμ H?o? s8%á 3 F ?
yNA?V F?×F X H?×Fé ì
=Q? s8% b?TN H?üe Cv ￥ F? ) ?
' Va1?? F ???"￥y￥I ?átá 3 F ) í F?yVr￥?aé? P s8%á 3? F 3 í F?b N
o3a?!e?F? a  F 3 í
 ? ? é￥ ) ?' ^cμ"￥y￥"￥×F0  L=?TV?? F 3 íμ H?o? s8%á 3 F ? A?V F?×F X H?×Fé ì
=Q? s8% b?TN H?üe Cv ￥ F? ) ?
' Va1?? F ???"￥y￥I ?átá 3 ?÷yát_? ?÷yát_? ?aé 3t????E ?aé 3t????E F ) í F?y￥k?
 F ) í F?y￥k?
 "￥×F0 "￥×F0 o? F? o? F? |×Fé ì |×Fé ì Q? Q? ?÷yát_? ?÷yát_? ?aé 3t????E ?aé 3t????E DNA2??aI ?y￥k?
 2??aI ?y￥k?
 ?? ?? ¨ Y ?? 3üe ¨ Y  ?? 3üe !′ !′ ? ? ?; ?; ^^?!?? 
?2%? ^^?!?? 
?2%? 80￥??%???  ￥??%???  D?·A?? D?·A?? !′a?2a?; !′a?2a?; ¨ o_? ? ` )8¨ò X Hüe ¨ o_?  ? ` )8¨ò X Hüe Y ?ê¨?D ?ê¨?D "S?a+s "S?a+s ?2?￥ DNA ?2?￥ ? ? ?÷yát_? ?÷yát_? ?aé 3t????E ?aé 3t????E ? dbrk?
 ? dbrk?
 ? i ? i M ? M ? + ? + ? ? ! ? !  "  " % ! % ! ! ? ! J r J r ?  ?  ×F DNA ×F ? y DNA ? y mRNA ?￥ mDNA ?￥ ?aé ?aé ?aé ?aé í%8?Jr"d
 ??4|ta??￡%4|t í%8?Jr"d
 ??4|ta??￡%4|t ?÷yát_? ?÷yát_? ?aé 3t2k?E ?aé 3t2k?E b  ?feê?k?
 b  ?feê?k? ?? ?? ¨%? +s? F8￥ Y ?? ¨%? +s? F8￥ Y  ?? !′ !′ D I 125 S:￥ IgG? D I S:￥ ? ?; ?; ^^?!?? 
?2%? ^^?!?? 
?2%? D I 125 S:￥ IgG? D I S:￥ ? !′a?2a?; !′a?2a?; ¨ o_? ? ` )8¨ò X Hüe ¨ o_?  ? ` )8¨ò X Hüe c F8￥ Y 3üe 3üe ?÷yát_? ?÷yát_? ?aé 3t2k?E ?aé 3t2k?E ?f??k?
 ?f??k?
 j??!?F?"S?a￥+s? F8 j??!?F?"S?a￥+s? F8 ?ap??A ?ap??A 37￥?! ü?!a
"￥×F0 ) ?M?s ?"S ü?!a
"￥×F0 ) ?M?s ?"S ?a
a?D+s? F8? 3 ?f?? ?a
a?D+s? F8? 3 ?f?? Q?
 ) ?????a?￥?ê Q?
 ) ?????a?￥?ê NEeL y ?
? 2 ù?
 F8h{ v NEeL y ?
? 2 ù?
 F8h{ v ?÷yát_? ?÷yát_? d??`?è￡E d??`?è￡E ?T??êk?￥"S?a;? ??? 3t?
?íC?￥ F 8 P¨
5 V?¨ d??` ?è￡é?Y {￥?ê ?T??êk?￥"S?a;? ??? 3t?
?íC?￥ F 8 P¨
5 V?¨ d??` ?è￡é?Y {￥?ê