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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)
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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 DNA-mediated transformation • Be familiar with PCR, RT PCR/Western Blotting, genomics and ELISA
What Is PCR? • DNA replication gone crazy in a test tube! • Makes millions of copies of a target sequence from template DNA • Uses heat-resistant Taq polymerase from Thermus aquaticus
3’ 5’ 5’ 3’ Forward primer Reverse primer 3’ 5’ 5’ 3’ Target sequence What Is Needed for PCR? • Template (the DNA you want to amplify for the study) • Sequence-specific primers flanking the target sequence: • Nucleotides (dATP, dCTP, dGTP, dTTP) • Magnesium ions (enzyme cofactor) • Buffer, containing salt • Taq polymerase
How Does PCR Work? • Heat (94°C) to denature DNA strands • 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’ 5’ 3’ Denaturation of DNA at 94°C 3’ 5’ 3’ 5’
5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ Annealing Primers • Primers bind to the template sequence • Taq polymerase binds to double-stranded substrate 3’ 5’ 3’ 5’ Primers anneal at 60°C
Taq Polymerase Extends… • Taq polymerase extends primer • DNA is replicated 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ Extends at 72°C 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’
Exact-length Target Product is Made in the Third Cycle Which means there’s extra DNA prior to cycle 3 Cycle 1 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ Cycle 2 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ Cycle 3 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’
Polymerase Chain Reaction
PCR Results • The PV92 Alu is dimorphic so there are two possible PCR products: 641 bp and 941 bp No insertion: 641 bp Alu insertion: 941 bp 300 bp Alu insert 641 bp 3’ 5’ Alu Amplified Region
Actual Alu PCR Results - +/- + 941 bp 641 bp - + +/-
DNA RNA Protein Trait Central Framework of Molecular Biology
Protein Size • Beta Lactamase • Ampicillin resistance • Green Fluorescent Protein (GFP) • Aequorea victoria jellyfish gene • araC regulator protein • Regulates GFP transcription
Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA
Why Perform Each Transformation Step? There’s lot’s of important stuff here… 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…
LB/Amp LB/Amp LB/Amp/Ara LB Grow?Glow? • 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 +pGLO
ara GFP Operon ara Operon araC GFP Gene araC B A D Effector(Arabinose) Effector (Arabinose) araC B A D araC GFP Gene RNA Polymerase RNA Polymerase B A D araC araC GFP Gene Gene Regulation
DNA RNA Protein Central Dogma of Molecular Biology DNA is composed of nucleotides. RNA is synthesized using DNA as the template in a process called Transcription. RNA is composed of nucleotides. Protein is synthesized from the information in RNA in a process called Translation. 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 5’ CCTGACTCCT GTGGAGAAGT CTGCCGTTAC • A ----------------> T DNA • GAG ----------------> GUG RNA • glutamate ----------------> valine protein Nucleotides: DNA: Adenine RNA: Adenine Guanine Guanine Cytosine Cytosine Thymine ---------------->Uracil Go BACK to figure 17.6 and convince yourself that this works. This will be a big part of the intro.
CCTNAGG -CC TNAGG- 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. Incubate with Bsu36I at 37C
CCTGTGG CCTGAGG Incubate with Bsu36I Incubate with Bsu36I Digestion of beta globin DNA with Bsu36I Normal beta globin gene (531 base pairs) Sickle beta globin gene (531 base pairs) + (531 base pairs) (331 base pairs) (200 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) SS: homozygous for sickle gene 500 bp 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
Heavy chain Disulfide bonds Light chain ELISA Antibody Structure
Applications • – Dipstick tests/ELISA • – Immunostaining • – Western blotting Real-world Applications of Antibodies Uses – Disease diagnosis – Basic Research – Immunotherapy Bio-Rad’s HIV-2 ELISA Kit Bio-Rad’s HIV Western Blot Kit
Students need to recognize molecular homologies/similarities A great FRQ from 2009
