文档介绍：第七章
常用分子生物学技术的原理及应用(二)
The popular technology in molecular biology: principle and application
Molecular Cloning
Vectors
plasmid
phage
cDNA
genomic libraries
cDNA libraries
DNA ligation
binant DNA molecules
Transformation
Selection
chromosome walking
molecular hybridization
DNA sequencing
The polymerase chain reaction (PCR)
inverse PCR
reverse transcriptase PCR
Quantitative PCR
asymmetric PCR
RNAi
Multiplex PCR
Southern Blotting
DNA fingerprinting
Western blotting
Northern blotting
Arbitrary primer PCR
fluorescence in situ hybridization (FISH)
Gene chip or DNA chip
上一章常用技术词汇
四核酸序列分析 Nucleic acid sequence analysis
核酸序列分析的基本原理
化学裂解法(Maxam-Gillbert法)---略
DNA链的末端合成终止法(Sanger法)
(Sanger双脱氧链终止法)
HO P O P O P O CH2
O
O
O
OH
OH
OH
O
A, C, G or T
1′
3 ′
OH
α
β
γ
5 ′
双脱氧核苷三磷酸
脱去
二、DNA链末端合成终止法
Sanger 1958年和1980年两次获Nobel奖
The Nobel Prize in Chemistry 1980
"for his fundamental studies of the biochemistry of nucleic acids, with particular regard to binant-DNA"
"for their contributions concerning the determination of base sequences in nucleic acids"
Paul Berg
Walter Gilbert
Frederick Sanger
1/2 of the prize
1/4 of the prize
1/4 of the prize
Stanford University
Stanford, CA, USA
Harvard University, Biological Laboratories
Cambridge, MA, USA
MRC Laboratory of Molecular Biology
Cambridge,
United Kingdom
1926-
1932-
1918-
三、DNA自动测序
用荧光替代放射性核素标记是实现DNA序列分析自动化的基础。
用不同荧光分子标记4种ddNTP,然后进行Sanger测序反应,产物经电泳(平板电泳或毛细管电泳)分离。
通过4种激光激发不同大小DNA片段上的荧光分子使之发射出4种不同波长荧光,检测器采集荧光信号,并依此确定DNA碱基的排列顺序。
ABI PRISM 310型 DNA测序仪
主要用于DNA序列分析(包括DNA测序,卫星序列、杂合子序列和三联体序列分析,单链构象多态分析,长片段PCR分析),遗传疾病诊断,亲子鉴定等