chap. 5. molecular tools for studying genes and gene activity
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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. - PowerPoint PPT PresentationTRANSCRIPT
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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
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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
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Fig. 5.6
Gel filtration chromatography
Autoradiography
densitometer
phosphorimaging
Molecular techniques
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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
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3. PCR
1. Taq DNA Polymerase
2.. dNTP + α-32P-dATP
A A
A A A
A
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5’3’ polymeriztion 5’3’ exonuclease 3’ 5’ exo.
4. Nick translation
5. Reverse transcription
Non-radioactive probe
Biotin, avidin, alkaline phosphatase
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Capillary transfer electrophoresis
hybridization
autoradiography
Southern blot
Southern blot
Northern blot
Western blot
In situ hybridization
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Fig. 5.13
DNA fingerprinting Minisatellite DNA
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DNA sequencing
1975, Sanger
ddNTP
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Next generation sequencing10 Mb to 1 Gb
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Restriction mapping
In vitro mutagenesis; site-directed mutagenesis with PCR protein engineering
GmATC ; DpnI
Reporter Gene Transcription ; lacZ, cat, luciferase