MCB 130L Lecture 1: DNA

Central Dogma of Molecular Biology This week: Molecular Biology Module Proposed by Francis Crick in 1958 Oversimplification b/c RNA can RT to DNA; RNA can replicate itself, noncoding RNAs, alternative splicing, introns, etc. Molecular biology is the study of the process of replication, transcription and translation of the genetic material Interesting fact: Crick did not have a PhD at the time of his discovery, switched from physics to mol biol after a WWII bomb destroyed his lab equipment. Proposed by Francis Crick, 1958

Recombinant DNA technology Creation of a novel combination (i.e. human and bacteria DNA) Applications: Cloning Sequencing Modification Mutagenesis Creation of novel fusion genes Cloning=making multiple copies of a fragment of DNA, i.e. gene Sequencing—If you’re interested in obtaining a DNA sequence, you can use recombinant DNA technology (i.e. PCR, cDNA/DNA library) to sequence that region Modification—mutagenesis: function of gene, necessity of gene fragment, amino acid, etc. fusion genes: improving activity of protein i.e. Maxygen gene shuffling technology ease of purification, fluorescent tagging, etc. cDNA, RNAi libraries

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 Gene structure—including transcriptional regulation Protein function—domains, phosphorylation/post-translational modifications, etc.

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

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 Plasmid=best example b/c can be amplified in bacteria E. Coli replication=20 minutes Can obtain huge amounts of DNA of interest Transition: PCR to amplify fragment of interest, then insert into plasmid to make recombinant DNA

Amplification of specific DNA sequences: Polymerase Chain Reaction (PCR) Applications: 1. general amplification 2. diagnostics 3. isolating DNA from ancient organisms 4. forensics Amplification for cloning, sequencing, basic research purposes Diagnostics for genetic diseases, infectious diseases, i.e. XDR TB Isolating DNA—Jurassic Park Forensics—identifying suspects/victims of criminal cases Invented by Kary Mullis (UCB PhD) while at Cetus Corp., Emeryville 1993 Nobel Prize in Chemistry

PCR movie

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! Contamination/sensitivity issues: especially when doing diagnostics, quantitative pcr, etc.

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 Taq withstands denaturing temperatures Pfu polymerase=proofreading

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

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 Plasmid=best example b/c can be amplified in bacteria E. Coli replication=20 minutes Can obtain huge amounts of DNA of interest Transition: PCR to amplify fragment of interest, then insert into plasmid to make recombinant DNA

Cloning movie

Cloning DNA: plasmid vectors Origin of replication Polylinker or multiple cloning site (MCS) Ampr gene (selectable) What would happen if any of these 3 features were missing? MCS: Nowhere to add the DNA of interest (Bacteriophages = alternative cloning vector)

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

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

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.

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

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 Plasmid=best example b/c can be amplified in bacteria E. Coli replication=20 minutes Can obtain huge amounts of DNA of interest Transition: PCR to amplify fragment of interest, then insert into plasmid to make recombinant DNA

Forming recombinant DNA molecules: ligation - T4 DNA ligase Requires ATP Phosphodiester bond Ligation of sticky ends is more efficient than blunt Ligation—creates phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another

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

Other Methods in recombinant DNA technology Southern blot DNA sequencing

Southern Blot Microarray technology evolved from Southern blotting

DNA Sequencing: dye terminator sequencing Dideoxynucleotide triphosphates/chain terminators Dye-terminator sequencing, capillary electrophoresis

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