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Biotechnology Learning Objectives • Be able to describe the components of DNA electrophoresis, and recognize patterns in a gel • Be able to describe the form and function of restriction enzymes (restriction endonucleases) • Be able to describe the process of DNA-mediated transformation of bacterial cells • Discuss the molecular basis for the results of a DNAmediated transformation • Be familiar with PCR, RT PCR/Western Blotting, genomics and ELISA • DNA replication gone crazy in a test tube! What Is PCR? • Makes millions of copies of a target sequence from template DNA • Uses heat-resistant Taq polymerase from Thermus aquaticus • Template (the DNA you want to amplify for the study) • Sequence-specific primers flanking the target sequence: 5’ 3’ What Is Needed for PCR? 3’ Forward primer 5’ 5’ 3’ Reverse primer 5’ 3’ Target sequence • Nucleotides (dATP, dCTP, dGTP, dTTP) • Magnesium ions (enzyme cofactor) • Buffer, containing salt • Taq polymerase • Heat (94°C) to denature DNA strands How Does PCR Work? • Cool (60°C) to anneal primers to template • Warm (72°C) to activate Taq polymerase, which extends primers and replicates DNA • Repeat multiple cycles Denaturing Template DNA Heat causes DNA strands to separate 5’ 3’ 3’ 5’ Denaturation of DNA at 94°C 5’ 3’ 3’ 5’ • Primers bind to the template sequence • Taq polymerase binds to double-stranded substrate 3’ 5’ Annealing 3’ 5’ Primers Primers anneal at 60°C 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ • Taq polymerase extends primer • DNA is replicated Taq Polymerase Extends… 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ Extends at 72°C 5’ 3’ 3’ 5’ 3’ 3’ 5’ 5’ Cycle 1 Exact-length Target Product is Made in the Third Cycle Which means there’s extra DNA prior to cycle 3 5’ 5’ 3’ 3’ 5’ 3’ 3’ 5’ Cycle 2 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ Cycle 3 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ Polymerase Chain Reaction No insertion: 641 bp • The PV92 Alu is dimorphic so there are two possible PCR PCR Results products: 641 bp and 941 bp 5’ Alu insertion: 941 bp 300 bp Alu insert 641 bp Alu Amplified Region 3’ Actual Alu PCR Results + 941 bp 641 bp + - +/- - +/- Central Framework of DNA Molecular RNA Biology Protein Trait • Beta Lactamase – Ampicillin resistance • Green Fluorescent Protein (GFP) – Aequorea victoria Protein jellyfishSize gene • araC regulator protein – Regulates GFP transcription Bacterial cell Bacterial DNA Plasmid DNA Genomic DNA There’s lot’s of important stuff here… Why Perform Each Transformation Step? Cell wall GFP 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression Beta-lactamase (ampicillin resistance) There’s an essential component that’s not shown… Grow? Glow? -pGLO • These are important Questions… • What’s the role of the LB (-pGLO) plate? • On which plates will colonies grow? – It depends on what you plated – What traits will be evident? – Why? • Which colonies will glow? – Why? -pGLO +pGLO +pGLO ara GFP Operon ara Operon ara C B A D araC Effector (Arabinose) Effector (Arabinose) Gene Regulation araC B A D araC RNA Polymerase araC B A D GFP Gene GFP Gene RNA Polymerase araC GFP Gene Central Dogma of Molecular Biology RNA is synthesized using DNA as the template in a process called Transcription. Protein is synthesized from the information in RNA in a process called Translation. DNA DNA is composed of nucleotides. RNA RNA is composed of nucleotides. Protein Protein is composed of amino acids. SS Sickle cell disease is caused by a single base mutation in the gene that codes for the beta globin subunit of hemoglobin. Normal beta globin gene 5’ CCTGACTCCT GAGGAGAAGT CTGCCGTTAC Sickle beta globin gene GTGGAGAAGT CTGCCGTTAC 5’ CCTGACTCCT A ----------------> T DNA GAG ----------------> GUG RNA glutamate ----------------> valine Nucleotides: DNA: Adenine Guanine Cytosine Thymine RNA: Adenine Guanine Cytosine ----------------> Uracil Go BACK to figure 17.6 and convince yourself that this works. This will be a big part of the intro. protein DNA can be analyzed by digestion with restriction enzymes Restriction enzymes are proteins that cut DNA at specific nucleotide sequences. The restriction enzyme Bsu36I cuts DNA with the sequence CC^TNAGG. CCTNAGG Incubate with Bsu36I at 37C -CC TNAGG- Digestion of beta globin DNA with Bsu36I Normal beta globin gene (531 base pairs) CCTGAGG Incubate with Bsu36I Sickle beta globin gene (531 base pairs) CCTGTGG Incubate with Bsu36I + (331 base pairs) (200 base pairs) (531 base pairs) Analysis of globin DNA by Gel Electrophoresis, following Bsu36I digestion _ DNA ladder AA uncut AA cut AS uncut AS cut SS uncut SS cut 1000 bp AA: homozygous for normal gene AS: heterozygous (trait) 500 bp SS: homozygous for sickle gene RFLP! + Types of Questions, students are likely to encounter (MCQ) • Reading a gel/Recognizing patterns • Applying concept of RFLP to genetic disorder, paternity cases • Linking experimentally derived genetic information to a cladogram • Describing process of transformation and describing its utility Biotech Extended… ELISA Antibody Structure Heavy chain Disulfide bonds Light chain ELISA EnzymeLinked Immunosorbant Assay ELISA Kit Results Uses – Disease diagnosis – Basic Research – Immunotherapy Applications – Dipstick tests/ELISA – Immunostaining – Western blotting Real-world Applications of Antibodies Bio-Rad’s HIV-2 ELISA Kit Bio-Rad’s HIV Western Blot Kit Example: Pregnancy Test More Biotech Extended… Students need to recognize molecular homologies/similarities A great FRQ from 2009