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

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.

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

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

  4. Methods of labeling DNA 1. kination; oligonucleotide 2. Gap Filling AATTC G 5’-GAATTC-3’ 3’-CTTAAG-5’ G CTTAA 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. 4. Nick translation 5’3’ polymeriztion 5’3’ exonuclease 3’ 5’ exo. 5. Reverse transcription Non-radioactive probe Biotin, avidin, alkaline phosphatase

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

  8. Fig. 5.13 Minisatellite DNA DNA fingerprinting

  9. DNA sequencing 1975, Sanger ddNTP

  10. Nextgeneration sequencing 10 Mb to 1 Gb

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

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