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()