核酸检测技术 ppt课件.ppt

文档介绍：第2章
核酸检测技术
核酸检测技术
直接对动植物有害生物基因序列和结构进行测定。
核酸的分离纯化
PCR技术
核酸杂交技术
精品资料
你怎么称呼老师？
如果老师最后没有总结一节课的重点的难点，你是否会认为老师的教学方法ent steps in each PCR cycle
PCR的扩增？
How does PCR work
The first cycle
The second cycle
How does PCR work
The third cycle
The fourth cycle
2. PCR的类型
多重PCR（multiplex PCR）
巢式PCR（nested PCR）
定量PCR（quantitative PCR）
不对称PCR（asymmetric PCR）
反向PCR（inverse PCR）
逆转录PCR（reverse transcription PCR）
Overlap PCR
… …
PCR using several primer pairs SIMULTANEOUSLY
Typically generates a product band for each primer pair
多重PCR（multiplex PCR）:What
多重PCR（multiplex PCR）
Multiplex PCR: Why
Detect several genes at once
eg. transgenic plant screen
Internal controls
VERY important
Tells you how well the PCR reaction worked
Reduces “false negatives”
Reduces “false positives”
多重PCR（multiplex PCR）
Multiplex PCR: How
Same as regular PCR
Care in primer design
Much greater chance of primer-dimers
Much greater chance of artifacts
Annealing temperatures must be close
多重PCR（multiplex PCR）
A Typical Multiplex PCR Reaction
Sterile Water ul
10X PCR Buffer ul
MgCl2 (50mM) ul
dNTP’s (10mM each) ul
Primer1FWD ul
Primer1REV ul
Primer2FWD ul
Primer2REV ul
Primer3FWD ul
Primer3REV ul
DNA Polymerase ul
DNA Template ul
Total Volume ul
多重PCR（multiplex PCR）
Multiplex PCR: Example
Three primer pairs
Control, resistance, and trait genes
Control gene fragment is largest and (almost) faintest
Trait gene is smallest and brightest
Resistance Gene
Trait Gene
Control Gene
Primers
多重PCR（multiplex PCR）
Multiplex PCR: Example
Three primer pairs
Resistance Gene
Trait Gene
Which are transgenic?
Control Gene
Primers
多重PCR（multiplex PCR）
Nested Multiplex PCR
2nd Stage PCR
Dilute 100 fold
Between 1st & 2nd stage reactions
1F
1R
2R
3R
5R
6R
3F
2F
6F
4F
5F
4R
Primary Multip