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Genetics
普通生物學
UNIT THREE
觀念11: DNA定序(DNA Sequencing)
端
單股DNA和帶有放射性
1. Sanger法(Sanger method)或Sanger雙去氧鏈終止法(Sanger
dideoxy chain termination method) :
1. 以5碳糖上第2和第3個碳均去氧的核苷酸為材料,合成的DNA之3',
不能再接新的核苷酸,造成DNA终止。
2. dsDNA加熱變性,快速冷卻成單股DNA,
(PP)標示的primer 配對。
3. 反應時分成四管:
a) #AZDNA+primer+DNA polymerase+4dNTPs+ddATP
b) ADNA+primer+DNA polymerase+4dNTPs+ddTTP
c) ADNA+primer+DNA polymerase+4dNTPs+ddGTP
d) 單股DNA+primer+DNA polymerase+4dNTPs+ddCTP
4. 終止反應,並用聚丙醯凝膠(polyacrylamide gel) 電泳分離。
5. 顯影後判讀X光片即可讀出DNA序列。
6. Frederick Sanger 因為這個方法獲得1980年諾貝爾獎。
Primer
5
OH
3
3'
-CTAAGCTCGACT
Template
dCTP, dGTP, DATP, DTTP
+ ddATP
- GATTCGAGCTGddA
GATTCGddA
F GddA
+ ddCTP
GATTCGAGddC
GATTddC
+ ddGTP
- GATTCGAGCTddG
GATTCGAddG
GATTCddG
ddG
+ ddTTP
- GATTCGAGCddT
-GATddT
- GAddT
G
3'
12
11
10
G
C
uuu9876543 21
5 As Tcc AccTT AG
Autoradiogram of
electrophoresis gel
Sequence of
complementary strand
500 1610
T天就在
下一秒就看到,

to sequence? We can calculate the probability that a base will not be
sequenced (P) using this equation:
P= e-m
where e is the base of the natural logarithm (e=2.72), and m is the num-
ber of sequenced bases divided by the total genome size. For example,
in the case of H. influenzae, with a genome size of 1.8 Mb, if research-
ers sequenced 9.0 Mb, m= 5 (that is, 9.0 Mb divided by 1.8 Mb):
Prem=e5 = 0.0067, or 0.67%.
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