Post on 15-Jan-2016
description
DNA DNADNA DNARNA DNARNA DNARNA DNARNA --
DNADNA3- 5DNA3cDNA53
isoschizomer (
DNA 200
50DNADNA hsd R--- hsd M--- hsd S---
DNADNAWArberHSmithDNathans197810
1.IhsdRhsdMhsdS 2.II 3.IIIhsdhsd
SAM5
ATPMg2+,SAM Mg+ATPMg2+ SAM 1000bp 2426lp
MMeselsonRYuan(1968)1970HOSmithKWWilcox(Haemophilus influenzae)Hind
1. II 2. I, II, III HindHaemophilus influenzae EcoRIEscherichia coliR
1. 4-6.. EcoRI 5-GAATTC-3 .
1 - 5.
2 -
3
isoschizomer 1. 2. 1 2 KpnI GGTAC C Asp718 G GTACC SstI CCGC GG SacI CCGC GG
isocaudamer BamH I GGATCC Bgl II AGATCT
1. DNA 2. 3. RFLP 4.
aDNA
b
20 uL1h1ug DNA 10mmol/LEDTA0.1%SDS)
A(EDTA)10mmolLMg2+ B(SDS)0.1(WV)() C6520min() AccBclBstNIBstXITaqI DDNA
12DNA3DNA45DNA6
DNA EDTASDSNaCl 1 g DNA 10U
DNAdam5-GATTC-3N6Bcl IMboIBamH IBgl IISau3A Idcm5-CCAGG -35-CCTTGG-3C5EcoR II5-CG-3C5
10TrisC1NaClKCIMg2+L(10mmolLNaCl)M(50mmolLNaCl)H(100mmolLNaCl)(DTT)BSADNA1NaCl23
DNADNA(DNA adenine methylase,Dam)GATC N6DNAA-N6
2. DNA(DNA cytosine methylase ,Dcm)CCAGGCCTGGCC5EcoR II
()II 1. HincII GTCGAC GTCAAC GTTGAC GTTAAC M.TaqITCGAAM.TaqIDNAGTCGACHincII
BamHI GGATCC
M.MspI m5CCGG
BamHICCGGM.MspIDNABamHI
DNAAluI(AGCT)HaeIII(GGCC)DNAM.EcoRIEcoRIEcoRI
2.TCGATCGAAGCTAGCT TaqI TaqI
M.TaqI
TCGA*TCGA* *AGCT*AGCT DpnI
3. DNA
m5CGm5CGm5CNGGm6ATC
C m5/4 CGG MspIHpaIIGm6ATC Sau3AI MobI
1RNARnase2DNADnase3RNA DNA
Rnase RNADNARNA 1. RNase A 10015min DNARNA RNA DNA-RNARNA 2. S7 RNAmRNA 3. RNase H DNA-RNARNAcDNARNAcDNA 4. RNase T1 3 RNase A
Dnase1. ds -DNA5-P; Ca2+ , Mg 2+ Ca2+ , Mn2+ ds -DNAMg 2+Mn2+
2. 1) DNA 2) RNADNA 3) 4)DNase IDNADNA
nick translation
S1 Zn 2+ (PH4.0-4.5).: DNA S1 RNA3233S1
S1
S1
S1
S1
IIIExoIII 1. E.coli28,000 kD 2. 1) 355- ds-DNApH 7.6-8.5 2) HDNA-RNARNApH 7.6-8.5 3) 3-3-3-OHDNApH 6.8-7.4 4) DNApH 7.6-8.5
3553
3. 1) ds-DNA 2) C>>A-T>>G 3) 5-5-ds-DNADNA 4. 1) DNA 2) ---- 3) DNA 5. 1) 2) ExoIIIGC(cDNA)
ExoIIIRNaseH
ds-DNA: , ,
DNADNA I Ecoli DNA polymerase I1. (109kDa) 53DNA: (ssDNA), 3 OH 5DsDNA Mg2+ dNTP
DNApolyI
53: dsDNA or DNA: RNA5dsDNA RNA:DNARNA(RNase H ) Mg2+
DNApolyI+ dNTP
353-OH dsDNA or ssDNA3-OHDNA53DNA Mg2+
DNApolyI Mg2+
DNApolyI+ dNTP+ dNTP
2. polI DNADNApoly
Mg2+, DNase IdNTP DNApoly I5--3dNTP,
cDNA
DNA
5---3Klenow
A*T Mg2+, DNApoly I [-P32]dATPT4 DNApolyT7 DNApoly
5- Mg2+
DNApolyI
5 -3 5 -3 3 -5 KlenowE.coli DNA polymerase I large fragment Klenow fragment1. , DNApoly I
2. 3 dNTP 3dNTP T4T73--5 A ,T4poly B3-- cDNA DNASanger T7TaqPCR
T4 DNA1.
Klenow 35100-1000 DNA
2. 3 dNTP dNTP() DNAdNTP dsDNAdNTP cDNA
3 dNTP 35
T7DNAT7DNApoly ,
/T4DNApolyKlenow 3 5Klenow1000
:A. T4B. C .
Taq DNA1. 94,00075 95352. DNA25DNA 4106 DNA DNA(95) DNA(45) (75)
Taq Pol PCR
1 DNARNase HDNA3 52 AMVM-MLVMoloney Reverse transcriptaseRNADNA
2 RNase H 1RNase H42C 42CpHpH 8.3 pH 7.6
3 cDNA 3 5DNA (DNApolyI, Klenow ) RT-PCR RNA
(Ligase) T4 DNA ligase T4 DNA or RNA() DNAPEG10% 150-200mM NaCl 12-16 ATP0.5mmol/L,5.0 mmol/L, 7.5 mmol/L.
DNA 53
(Ligase)
(Ligase)
(Ligase)
1) 2)
Weiss 3720min1 nmol 32p[, -32P]ATP NEB (New England Biolabs)
20l, 16,30 min,300g/ml(0.12M 5) / HindIII50%DNA
1 Weiss= 67 (NEB ) (Molecular cloning 60 NEB unit) 1 NEB= 0.015 Weiss 16 4hr 4 overnight ATPDNA
()E.coli DNA
T4DNA ligase,-(NAD+)
cDNARNADNA,RNA
(Ligase)
(Ligase)
CIPCIAP BAPDNA or RNA 5DNA55BAPCIPK5mM EDTA 65 1hr ( 75 10min),: Alkaline Phosphatase Calf Intestinal Mucosa
DNADNA
Terminal transferase,DNApoly
dNTPDNA3
T, C Co2+ A, GMg2+
1. DNA, 3, 3,3DNA 2. cDNA3 ddNTP()