DNA????????; RNA?????????

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?3? ???????
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? ????
1???
DNA???????? RNA?????????
2????
????,???????????????????DNA???????,RNA????
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???????
3?????
????????????,?????????,??????????????????pH????
?
???????????,??pI??,???????????
????????????????????????????,?????????????
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???????
? ??????
???????????,??????????260nm????????????DNA?RNA,
??????A260?????????
A260/ A280???????????
??DNAA260/ A280 1.8 ??RNAA260/ A280 2.0
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???????
? ?????
????????????????,?????????,???????????????????
????????????????????????????

1.????
?????,?260nm???????,??????(hyperchromic
effect)???????????DNA??????

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DNA????( DNA denaturation )
? D.S. DNA
S.S. DNA
( ??, ??pH, ??, ?? )
???????
? S.S. DNA????
? S.S. DNA ??????
? S.S. DNA???A 260 nm UV ??? (
Hyperchromicity )
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???????
? ?????
2. ????
?????????????(????)??(?????????)??????DNA?????
???(Tm)?
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???????
????? (????)
?DNA?????????,A260?????????
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Concentration 50 ?g/mL

dNTPs A260 1.60
S.S DNA A260 1.37
1.185
D.S DNA A260 1
?
Tm
OD????????(???85-95?)
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???????
? ?????
Tm??DNA?????G-C??????????pH??????
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Marmur-Doty Formula
Tm 69.3 0.41 GC
what is meaning ?
Evaluation GC of DNA
?
Tm1 lt Tm2
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l    ????????? ( Jump of Hyperchromicity )
?????DNA?????????, ??AT?????? ??, ??????,
???????????, ???????.
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? ?? Tm????
? ? A, T, C, G ????????
GC?? ? Tm???
GC?? ? Tm ???
? GC????????
AT??????,????,Tm ??
?????Tm???????(??????)
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?????Tm???????
gtgt
Tm
ltlt
37.6 ? 57 ?
????? ? ?????????
????,??? D.S DNA?????AT???
???(promoter, terminator region )
???????????? DNA????
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? ???D.S DNA??????
?????Tm??????, ?????????
? ??100bp ? D.S DNA????
????, ????,Tm???
? ????????,???
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? ??????
??DNA???????
????????
????DNA???
Na????????????? ???????????
??????
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Tm ?
Tm ?
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???pH?????
pH 12
?? ? ???
???????????
NH2 ? NH3 (???)
pH 2-3
??????, ???????? ???Tm ???
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???????
? ?????
1.?? (renaturation)
DNA????????,??????????????,??????????????????
???????
?????????????????,??????????????
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DNA ????? (anneal or renaturation)
Denaturation ?
D.S DNA
S.S DNA
? Renaturation
????????????????? (
Depends on the collision of complementary S.S.
DNA )
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???????
? ?????
2. ????(hypochromic effect)
????DNA??????????????,????A260????,???????????
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??DNA???????
? S.S, DNA ????? C0
? DNA ???, dNt ????? (????, ????)
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?????????
?   ???????S.S. DNA, ??????????? ????????
? ??DNA????? ??( randomly collision )
??second order kinetics formula (???????)
dCt / dt -KCt2
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dCt / dt -KCt2 ??
Ct / C0 1 / 1KC0t
? Ct / C0 1/2 ?
Ct / C0 1/2 1 / 1 KC0t(1/2)
K 1 / Cot(1/2)
Cot(1/2) 1/K (mol. Sec / L)
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Cot(1/2) 1/K (mol. Sec / L)
? ???DNA Cot ½ ????, Cot ½???K???,??DNA??????
? K??????????????????DNA????? ?
?????????,?K????X???? K1/X, ??, Cot ½
1/K X
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Cot(1/2) 1/K (mol. Sec / L)
? ???(X)----DNA???????????????? e.g. (A)n,
X 1 (AT)n, X 2 (AGC)n, X 3
(AGCT)n, X 4 (AGCTG)n, X 5 ? Cot ½
??DNA????????
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???????
? ?????
3. ???? (hybridization)

• ???????DNA???DNA
• RNA? ??DNA???
• ???20 bp??????
• ???????????
• ????????????

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???????
? ?????
3. ???? (hybridization)
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???????
? ?????
3. ???? (hybridization)
DNA?????,????????????DNA??,????????????DNA-DNA
?DNA-RNA??????
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????
D.S. DNA
? S.S. DNA
S.S. DNA ( as probe)
p32 or biotin labeled
Column absorb D.S DNA
X-film
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??
??????
Absorb D.S. DNA
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(No Transcript)
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1975 E. M. Southern
Southern blot
S.S. DNA S.S. DNA
1977.    J.C. Alwine
S.S. RNA S.S. DNA
Northern blot
1978.    Hany Towbin
Western blot
Protein antibody
?????? (In situ hybridization )
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(No Transcript)
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? DNA ? ? ? ? ? ? (DNA
sequencing )
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1. ??
1972. Herbert Boyer
????????II (RE-II)

• Sanger C.
• , ?

• Maxam Gilbert
• ?????

• Sanger C.
• ddNTP(?)?

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2. ??
(1) Chemical sequencing
G?? pH 8.0 ????? ? G ? m7G ?C8N9??
??G
AG??pH 2.0 ????(pidine)? ??? N???? ???
C?? 1.5 mol/L NaCl C ?(hydrazine)?
?C
T C?? ?(hydrazine) ? ????? ???5C??????
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Chemical sequencing
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(2) ????
4ddNTP??? DNA???? ?ddNTP(??????)
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? DNA????(Methylation) (m5C in
Eukaryote)
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1. DNA?m5C???
CH3
m5C
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2. ??????
???????Am ?????m22,7G
??
. a. DNA m5dC
5-?????? m6dA N6-?????? n2dA
2-???????
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b. RNA m1A 1-???? m6A N6-????
m26A N6, N6-????? m6AmN6, 2'-O-?????
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3 Function of m5C

• ????,????,????,???????????m5C????(5-?????????
  ?)

• m5C---?????????????

• m5C-----?Z-DNA?????????

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? m5C ????????
C ? m5C ? T ? gene mutation (??)
O
CH3
????
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????