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MCB 130L

MCB 130L. Lecture 1: DNA. Central Dogma of Molecular Biology. Proposed by Francis Crick, 1958. Recombinant DNA technology. Recombinant DNA: Creation of a novel combination (i.e. human and bacteria DNA) Applications: Cloning Sequencing Modification Mutagenesis

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MCB 130L

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  1. MCB 130L Lecture 1: DNA

  2. Central Dogma of Molecular Biology Proposed by Francis Crick, 1958

  3. Recombinant DNA technology Recombinant DNA: Creation of a novel combination (i.e. human and bacteria DNA) Applications: • Cloning • Sequencing • Modification • Mutagenesis • Creation of novel fusion genes

  4. Importance of recombinant DNA • Basic research • Gene structure • splicing • transcriptional regulation • Protein function • domain structure • post-translational modifications • phosphorylation sites • Biotechnology • Insulin, growth hormone • Gene shuffling • Gene therapy

  5. Experimental Proposal:To determine the role of HexB in immune defense against tuberculosis? Recombinant DNA technology Clone HexB Recombinant DNA technology Clone HexB Mutate HexB Knockout/overexpress HexB Purify Protein Biochemical assays Protein-protein interaction X-ray crystallography Antibody production phenotype domain characterization phenotype cellular localization

  6. Essential steps in the generation of recombinant DNA DNA (genomic, plasmid, PCR, ….) DNA fragmentation/digestion DNA Separation and purification Forming recombinant DNA: ligation Cloning DNA: Transformation, selection and amplification

  7. Amplification of specific DNA sequences: Polymerase Chain Reaction (PCR) Applications: 1. general amplification 2. diagnostics 3. isolating DNA from ancient organisms 4. forensics Invented by Kary Mullis (UCB PhD) while at Cetus Corp., Emeryville 1993 Nobel Prize in Chemistry

  8. PCR movie

  9. Amplification of specific DNA sequences: Polymerase Chain Reaction (PCR) 1. Logarithmic amplification: # of copies = 2n, n = # of cycles 2. Sensitive: a single molecule can be amplified 3. Contamination a problem!

  10. Amplification of specific DNA sequences: Polymerase Chain Reaction (PCR) 1. Taq DNA polymerase from thermophilic bacteria (Thermus aquaticus, error rate 1/105) 2. dNTPs (dATP, dCTP, dTTP, dGTP) 3. Template = DNA to be amplified 4. Primers: 18-20 nucleotides complementary to template 5. Temperature cycling: 20-30 cycles Denaturation 95ºC Annealing 55ºC to 60ºC Extension 72ºC

  11. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ Amplification of specific DNA sequences: Polymerase Chain Reaction (PCR) 72ºC (Polymerase optimal temperature) 95ºC (Denaturation) 55ºC (Annealing) Cycle 1 (same procedure will be repeated 20-30 times)

  12. Essential steps in the generation of recombinant DNA DNA (genomic, plasmid, PCR, ….) DNA fragmentation/digestion DNA Separation and purification Forming recombinant DNA: ligation Cloning DNA: Transformation, selection and amplification

  13. Cloning movie

  14. Cloning DNA: plasmid vectors Origin of replication Polylinker or multiple cloning site (MCS) Ampr gene (selectable) (Bacteriophages = alternative cloning vector)

  15. Multiple cloning site Region of plasmid containing multiple restriction enzyme sites to enable insertion of DNA of interest

  16. Cutting DNA: restriction enzymes Site specific endonucleases produced by bacteria Recognize palindromic sequences (same 5’ --> 3’ on both strands) Evolved to cleave bacteriophage DNA

  17. Cutting DNA: restriction enzymes How do bacteria survive with restriction enzyme that cleaves DNA? - bacteria DNA is protected from cleavage by methylation Figure 4: Bacteria cells that produce restriction endonucleases also produce modification enzymes that methylate bases in the recognition site.

  18. Separating and purifying DNA fragments: gel electrophoresis • DNA is negatively charged • Moves to the (+) pole in electric field • Ethidium bromide intercalates DNA, fluoresces in UV light

  19. Essential steps in the generation of recombinant DNA DNA (genomic, plasmid, PCR, ….) DNA fragmentation/digestion DNA Separation and purification Forming recombinant DNA: ligation Cloning DNA: Transformation, selection and amplification

  20. Forming recombinant DNA molecules: ligation • - T4 DNA ligase • Requires ATP • Phosphodiester bond • Ligation of sticky ends is • more efficient than blunt

  21. Cloning DNA molecules: transformation, selection and amplification • Transformation = Introduction of plasmid into bacteria • Make “competent” bacteria • Add DNA • Inefficient uptake • Selection for antibiotic resistance • Amplification: Bacteria replicate w/ plasmid

  22. Other Methods in recombinant DNA technology • Southern blot • DNA sequencing

  23. Southern Blot Microarray technology evolved from Southern blotting

  24. DNA Sequencing: dye terminator sequencing

  25. This week’s lab: • PCR • Restriction Digests • Agarose Gel Electrophoresis

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