1. Pulsed-field gel electrophoresis
Chap. 5. Molecular tools for studying genes and gene activity
Gel electrophoresis
Log MW
Agarose
polyacrylamide
1. Size
2. Purification
3. concentration
Migration distance (cm)
Pulsed-field gel electrophoresis
Separate ; 2 Mb ~ 0.2 Mb

2. SDS-polyacrylamide gel electrophoresis
Roles of SDS
Denaturation
Coat negative charge
Pre-stained size marker
Staining
DNA ; Ethidium Bromice (EtBr)
Protein ; coomassie brilliant blue

3. Gel filtration chromatography
Fig. 5.6
Molecular techniques
Gel filtration chromatography
Autoradiography
densitometer
phosphorimaging

4. Methods of labeling DNA 1. kination; oligonucleotide
2. Gap Filling
5’-GAATTC-3’
3’-CTTAAG-5’ G
CTTAA
AATTC G
1. DNA Polymerase
2.. α-32P-dATP, TTP
GAATT
CTTAA

5. 3. PCR
1. Taq DNA Polymerase
2.. dNTP + α-32P-dATP A A A A A A

6. 5. Reverse transcription
4. Nick translation
5’3’ polymeriztion
5’3’ exonuclease
3’ 5’ exo.
5. Reverse transcription
Non-radioactive probe
Biotin, avidin, alkaline phosphatase

7. Capillary transfer Southern blot electrophoresis hybridization
autoradiography
Southern blot
Northern blot
Western blot
In situ hybridization

9. Fig. 5.13
Minisatellite DNA
DNA fingerprinting

10. DNA sequencing
1975, Sanger
ddNTP

11. Next generation sequencing
10 Mb to 1 Gb

12. In vitro mutagenesis; site-directed mutagenesis with PCR
Restriction mapping
In vitro mutagenesis; site-directed mutagenesis with PCR
 protein engineering
GmATC ; DpnI
Reporter Gene Transcription ; lacZ, cat, luciferase