Chapter 11. Studying gene expression and function

1

• Chapter 11. Studying gene expression and function

2
??? ??? ????
3
?????? ??? ?? ??? ?? - ?? ??? ??? ??
???? ?? - Cytochrome C polynucleotide? ?? -
Electro-dense Acetate ?? - ?? - ?? ??
4
???? ???? ?? ?? 1. ?? ?????? ??? ???? DNA-RNA
??? - Intron? ? ? ??? ??
5
2. ??????? ??? ?? DNA-RNA ??? ?? - S1
nuclease ?? ?? ??? ???? ???? ?? ???? -
Alkali ?? RNA ??
6
(No Transcript)
7
3. Primer extension - RNA? 5 ??? ?? - Primer
???? ???? ??? ? - Reverse transcriptase
8
RNA ??? ??? ?? ?? ??? 1. Northern
hybridization - Formaldehyde gel - ??? - ???
?? ????? - ???? ??? ?? ??? - ????
ribosomal RNA 4718bp 1874bp
9
2. ??? PCR (RT-PCR) - Reverse transcriptase -
Taq. Polymerase - ???? ?? ?? ?? - RACE -
Rapid amplification of cDNA ends - RNA ???
5 ? 3 ??? ??
10
??? ??? ??? ?? ?? - ?? ??? (?? ??)
11
DNA ???? ??? ???? ?? 1. DNA-protein ???? gel
retardation - ???? ?? ?? ??? - ???? 10bp ??
12
Gel retardation experiment
13
2. Footprinting with DNase I - ?? ???? DNA ??
?????? ????? Dnase I? ?? Endonuclease? ?? ????
???? ??
14
DNase I footprinting - ? DNA ?? ? ? ??? ???
????? ?? - ?? ??? ?? - DNA ??? ??? ???? ?? ???
DNase I ?? - Phosphodiester bond? ??
15
3. Modification interference assays - ?? ???? ??
???? ?? ? ?? DNA? ???? ??.
16
Modification interference assay -
End-labelling - Dimethyl sulphate Methyl
groups to guanine NT - Piperidine ???? NT?
DNA ??? ??
17
Deletion analysis? ??? ????? ??
18
Reporter gene - ?????? ???? ?? ??? - ?????
?? - ????? ?? ??
19
(No Transcript)
20
Deletion analysis - ??? ?? ?? - ?? ??? ?? - ???
??? ?? ???? ??
21
HRT Hybrid-release translation HART
Hybrid-arrest translation - ???? ?? ???? ????
?? - ?? ?? ??? reticulocyte ??? - 35S-methionine
19 amino acid
22
HRT Hybrid-release translation - Specific
cDNA - ?? - Nitrocellulose or nylon membrane -
mRNA sample - Hybridized mRNA - Translation
23
Hybrid-arrest translation - mRNA single
strand cDNA - Translation
24
Analysis of proteins by in vitro mutagenesis
25
???? ?? 1. ???? ????? ?? 2. ???? ???????
NT ?? - Endonuclease, Bal31 3.
Oligonucleotide ?? - ???? - ?-helix
(??? ??)
26
Oligonucleotide-directed mutagenesis (point
mutation) - Single-stranded DNA M13 vector -
Double-stranded DNA - Short primer
(oligonucleotide) - Klenow fragment
- dNTPs - Transfection into E. coli -
Semi-conservative nature of DNA replication
27
???? ???? ????? ?? ?? 1. Phage display -
Phage display library - Microtitre well or
Chromatography column
28
???? ???? ????? ?? ?? 2. Yeast two-hybrid
system - Bait vector - Prey vector (library)