第二章 基因工程中的工具酶
DESCRIPTION
第二章 基因工程中的工具酶. 限制性内切酶 — 主 要用于 DNA 分子的特异切割 DNA 甲基化酶 — 用 于 DNA 分子的甲基化 核酸酶 — 用 于 DNA 和 RNA 的非特异性切割 核酸聚合酶 — 用 于 DNA 和 RNA 的合成 核酸连接酶 — 用 于 DNA 和 RNA 的连接 核酸末端修饰酶 — 用 于 DNA 和 RNA 的末端修饰 其它酶类 -- 用于生物细胞的破壁,转化,核酸纯化,检测等. 常用的工具酶:. 一、限制性内切酶. (一)限制修饰系统的种类 (二)限制性内切酶的定义、命名 (三)限制酶的特点 - PowerPoint PPT PresentationTRANSCRIPT
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DNA DNADNA DNARNA DNARNA DNARNA DNARNA --
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DNADNA3- 5DNA3cDNA53
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isoschizomer (
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DNA 200
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50DNADNA hsd R--- hsd M--- hsd S---
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DNADNAWArberHSmithDNathans197810
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1.IhsdRhsdMhsdS 2.II 3.IIIhsdhsd
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SAM5
ATPMg2+,SAM Mg+ATPMg2+ SAM 1000bp 2426lp
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MMeselsonRYuan(1968)1970HOSmithKWWilcox(Haemophilus influenzae)Hind
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1. II 2. I, II, III HindHaemophilus influenzae EcoRIEscherichia coliR
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1. 4-6.. EcoRI 5-GAATTC-3 .
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1 - 5.
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2 -
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3
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isoschizomer 1. 2. 1 2 KpnI GGTAC C Asp718 G GTACC SstI CCGC GG SacI CCGC GG
isocaudamer BamH I GGATCC Bgl II AGATCT
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1. DNA 2. 3. RFLP 4.
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aDNA
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b
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20 uL1h1ug DNA 10mmol/LEDTA0.1%SDS)
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A(EDTA)10mmolLMg2+ B(SDS)0.1(WV)() C6520min() AccBclBstNIBstXITaqI DDNA
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12DNA3DNA45DNA6
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DNA EDTASDSNaCl 1 g DNA 10U
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DNAdam5-GATTC-3N6Bcl IMboIBamH IBgl IISau3A Idcm5-CCAGG -35-CCTTGG-3C5EcoR II5-CG-3C5
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10TrisC1NaClKCIMg2+L(10mmolLNaCl)M(50mmolLNaCl)H(100mmolLNaCl)(DTT)BSADNA1NaCl23
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DNADNA(DNA adenine methylase,Dam)GATC N6DNAA-N6
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2. DNA(DNA cytosine methylase ,Dcm)CCAGGCCTGGCC5EcoR II
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()II 1. HincII GTCGAC GTCAAC GTTGAC GTTAAC M.TaqITCGAAM.TaqIDNAGTCGACHincII
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BamHI GGATCC
M.MspI m5CCGG
BamHICCGGM.MspIDNABamHI
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DNAAluI(AGCT)HaeIII(GGCC)DNAM.EcoRIEcoRIEcoRI
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2.TCGATCGAAGCTAGCT TaqI TaqI
M.TaqI
TCGA*TCGA* *AGCT*AGCT DpnI
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3. DNA
m5CGm5CGm5CNGGm6ATC
C m5/4 CGG MspIHpaIIGm6ATC Sau3AI MobI
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1RNARnase2DNADnase3RNA DNA
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Rnase RNADNARNA 1. RNase A 10015min DNARNA RNA DNA-RNARNA 2. S7 RNAmRNA 3. RNase H DNA-RNARNAcDNARNAcDNA 4. RNase T1 3 RNase A
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Dnase1. ds -DNA5-P; Ca2+ , Mg 2+ Ca2+ , Mn2+ ds -DNAMg 2+Mn2+
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2. 1) DNA 2) RNADNA 3) 4)DNase IDNADNA
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nick translation
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S1 Zn 2+ (PH4.0-4.5).: DNA S1 RNA3233S1
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S1
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S1
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S1
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S1
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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
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3553
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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)
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ExoIIIRNaseH
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ds-DNA: , ,
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DNADNA I Ecoli DNA polymerase I1. (109kDa) 53DNA: (ssDNA), 3 OH 5DsDNA Mg2+ dNTP
DNApolyI
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53: dsDNA or DNA: RNA5dsDNA RNA:DNARNA(RNase H ) Mg2+
DNApolyI+ dNTP
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353-OH dsDNA or ssDNA3-OHDNA53DNA Mg2+
DNApolyI Mg2+
DNApolyI+ dNTP+ dNTP
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2. polI DNADNApoly
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Mg2+, DNase IdNTP DNApoly I5--3dNTP,
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cDNA
DNA
5---3Klenow
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A*T Mg2+, DNApoly I [-P32]dATPT4 DNApolyT7 DNApoly
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5- Mg2+
DNApolyI
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5 -3 5 -3 3 -5 KlenowE.coli DNA polymerase I large fragment Klenow fragment1. , DNApoly I
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2. 3 dNTP 3dNTP T4T73--5 A ,T4poly B3-- cDNA DNASanger T7TaqPCR
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T4 DNA1.
Klenow 35100-1000 DNA
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2. 3 dNTP dNTP() DNAdNTP dsDNAdNTP cDNA
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3 dNTP 35
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T7DNAT7DNApoly ,
/T4DNApolyKlenow 3 5Klenow1000
:A. T4B. C .
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Taq DNA1. 94,00075 95352. DNA25DNA 4106 DNA DNA(95) DNA(45) (75)
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Taq Pol PCR
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1 DNARNase HDNA3 52 AMVM-MLVMoloney Reverse transcriptaseRNADNA
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2 RNase H 1RNase H42C 42CpHpH 8.3 pH 7.6
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3 cDNA 3 5DNA (DNApolyI, Klenow ) RT-PCR RNA
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(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.
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DNA 53
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(Ligase)
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(Ligase)
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(Ligase)
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1) 2)
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Weiss 3720min1 nmol 32p[, -32P]ATP NEB (New England Biolabs)
20l, 16,30 min,300g/ml(0.12M 5) / HindIII50%DNA
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1 Weiss= 67 (NEB ) (Molecular cloning 60 NEB unit) 1 NEB= 0.015 Weiss 16 4hr 4 overnight ATPDNA
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()E.coli DNA
T4DNA ligase,-(NAD+)
cDNARNADNA,RNA
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(Ligase)
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(Ligase)
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CIPCIAP BAPDNA or RNA 5DNA55BAPCIPK5mM EDTA 65 1hr ( 75 10min),: Alkaline Phosphatase Calf Intestinal Mucosa
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DNADNA
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Terminal transferase,DNApoly
dNTPDNA3
T, C Co2+ A, GMg2+
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1. DNA, 3, 3,3DNA 2. cDNA3 ddNTP()