Chap. 5. Molecular tools for studying genes and gene activity

Gel electrophoresis

Agarosepolyacrylamide

1. Size2. Purification3. concentration

Log

MW

Migration distance (cm)

Separate ; 2 Mb ~ 0.2 Mb

Pulsed-field gel electrophoresis

SDS-polyacrylamide gel electrophoresis

Roles of SDS

1. Denaturation2. Coat negative charge

Staining-DNA ; Ethidium Bromice (EtBr)-Protein ; coomassie brilliant blue

Pre-stained size marker

Fig. 5.6

Gel filtration chromatography

Autoradiography

densitometer

phosphorimaging

Molecular techniques

Methods of labeling DNA1. kination; oligonucleotide

5’-GAATTC-3’3’-CTTAAG-5’

GCTTAA

AATTCG

2. Gap Filling

GAATTCTTAA

1. DNA Polymerase

2.. α-32P-dATP, TTP

3. PCR

1. Taq DNA Polymerase

2.. dNTP + α-32P-dATP

A A

A A A

A

5’3’ polymeriztion 5’3’ exonuclease 3’ 5’ exo.

4. Nick translation

5. Reverse transcription

Non-radioactive probe

Biotin, avidin, alkaline phosphatase

Capillary transfer electrophoresis

hybridization

autoradiography

Southern blot

Southern blot

Northern blot

Western blot

In situ hybridization

Fig. 5.13

DNA fingerprinting Minisatellite DNA

DNA sequencing

1975, Sanger

ddNTP

Next generation sequencing10 Mb to 1 Gb

Restriction mapping

In vitro mutagenesis; site-directed mutagenesis with PCR protein engineering

GmATC ; DpnI

Reporter Gene Transcription ; lacZ, cat, luciferase