第六章：DNA测序

ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
A C G T
A: dNTP + ddATP C: dNTP + ddCTP G: dNTP + ddGTP T: dNTP + ddTTP
2. Related strategy
(1) Labeling • Incorporation
• Chemical reaction
• Cleavages at specific bases • Running PAGE (7M urea) 6 % ~ 20% • Autoradiography
A>C G
T+C
C
A A T G G C G C C C A C T T C
Hale Waihona Puke 4. Notes• specificity • length (200-250bp )
Brief protocols
• Denature templates (ssDNA or ds DNA)
• Annealing • Labeling • Termination • Running gel
65 C to <35 C > 15~30min RT 2~5min 37C 5min,stop solution 75C 2min, running at 55 C, 2 loading 100C 2min
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CATC CATC CATC CATC CATC CATC CATC dITP
ddATP, ddCTP, ddGTP, ddTTP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CATCGTA CATCGTAC CATCGTAC CATCGTAC CATCGTAC CATCGTAC CATCGTAC dITP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CATCGTA CATCGTAC CATCGTACG CATCGTACGT CATCGTACGT CATCGTACGT CATCGTACGT dITP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CATCGT CATCGT CATCGT CATCGT CATCGT CATCGT CATCGT dITP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CATCGTA CATCGTA CATCGTA CATCGTA CATCGTA CATCGTA CATCGTA dITP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CATCGTA CATCGTAC CATCGTACG CATCGTACGT CATCGTACGTA CATCGTACGTAG CATCGTACGTAGC dITP
when a ddNTP is integrated
P
OH
OH
P
OH
H
P
H
H
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’
dITP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’
[-32P] dATP , [-33P] dATP, [-35S] dATP
• End-labeled primers
[ -32P]rATP
(2) Template
• ss DNA prepared from M13mp & phagemid • ds DNA plasmid
(3) Primer
stop solution
75C 2min, running at 55 C, 2 loading
(2) PCR reaction
(2) PCR reaction
CircumVent Thermal Cycle Dideoxy DNA Sequencing Kit
with
Vent R (exo-) DNA Polymerase
3. Procedure
• Full manual operation
• PCR reaction • Automatic sequencing
(1) Full manual operation
United States Biochemical, USB
Sequenase Version 2.0 DNA Sequencing Kit
Brief protocols
• Denature templates
• Annealing • Labeling • Termination • Running gel
(ssDNA or ds DNA) 100C 2min
15~30min
65 C to <35 C RT 2~5min 37C 5min
-complementation
M13mp18/19, pUC18/19, pGEM series, pBluescript, pTZ series
- 40
- 20
U
U
E1 S K….…....S H3
MCS
R R
- 20 - 40
-complementation
M13mp18/19, pUC18/19, pGEM series, pBluescript, pTZ series
dITP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ C C C C C C C dITP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CATCGTA CATCGTAC CATCGTACG CATCGTACGT CATCGTACGTA CATCGTACGTAG CATCGTACGTAG dITP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CATCGTA CATCGTAC CATCGTACG CATCGTACG CATCGTACG CATCGTACG CATCGTACG dITP
- 40
- 20
U
U
T7
E1 S K….…....S H3
MCS
T3
R R
- 20 - 40
-complementation
M13mp18/19, pUC18/19, pGEM series, pBluescript, pTZ series
Lane A: dNTP + ddATP Lane C: dNTP + ddCTP Lane G: dNTP + ddGTP Lane T: dNTP + ddTTP 2 loading
• size, G+C, Tm, degeneration, 2nd structure • position • universal primers
Universal(Forward), Reverse, T7, T3, SP6 etc
- 20
U
E1 S K….…....S H3
MCS
R
- 20
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CATCGTA CATCGTAC CATCGTACG CATCGTACGT CATCGTACGTA CATCGTACGTA CATCGTACGTA dITP
DNA Sequencing
DNA 序列测定
第一节 Maxam-Gilbert DNA 化学降解法
一、基本原理 直接或间接特异性识别4 种碱基
特定化学试剂可对碱基进行特异性修饰 在修饰碱基处(5‘或3’)打断磷酸二酯键
Reagents for MG DNA sequencing
Base specificity
G
Base reaction
dimethyl sulphate (硫酸二甲酯 ), methylation
A+G
C+T C A>C
acid (哌啶甲酸), pH2.0， 脱嘌呤
hydrazine (肼)，打开嘧啶环 hydrazine + NaCl (1.5M) 90C, NaOH (1.2M)， 断裂反应
PCR
PCR procedure
95 C 20 sec 55 C 20 sec 72 C 20 sec 20 cycles
T G C A
G C A T T A T A G A A G T* A T C A A T T T
Primer extension for CTC gene
T T T A A G T G A A A A A A C A G C T* T A A A T T C C A A A A
哌啶（90 C, 1M) 在修饰位点两端使DNA 的糖-磷酸链发生裂解
A>C G
T+C
C
A A T G G C G C C C A C T T C
2. Procedure
• Templates
a defined DNA restriction fragment radioactively labeled at one end with 32P
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CA CA CA CA CA CA CA dITP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CAT CAT CAT CAT CAT CAT CAT dITP
Core principle Key technique
dATP, dCTP, dGTP, dTTP
Template 3’AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5’ CATCG CATCG CATCG CATCG CATCG CATCG CATCG dITP
ddATP, ddCTP, ddGTP, ddTTP
dATP, dCTP, dGTP, dTTP
• it is better to read Maxam and Gilbert’s article before action
第二节 Sanger dideoxy-mediated chain-termination method
1. Core principle
DNA synthesis will be terminated
(3) Automatic sequencing ABI PRISM™ dRhodamine Terminator Cycle Sequencing Ready Reaction Kit
With AmpliTaq® DNA Polymerase, FS
PE Applied Biosystems a division of Perkin-Elmer