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

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

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

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

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

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

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

Fig. 5.13 Minisatellite DNA DNA fingerprinting

DNA sequencing 1975, Sanger ddNTP

Next generation sequencing 10 Mb to 1 Gb

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