Topic 14 - Molecular Techniques 43fc03c318774edbbf34cf34c949b6b6.pdf

💻 Topic 14 - Molecular Techniques CBG - Group work Session 14.docx 14.1 - Molecular Techniques What are 2 ways in which DNA can be prepared and stored Deactivate DNA damaging enzymes by heat/remo...

💻 Topic 14 - Molecular Techniques CBG - Group work Session 14.docx 14.1 - Molecular Techniques What are 2 ways in which DNA can be prepared and stored Deactivate DNA damaging enzymes by heat/removal of divalent cations cofactors by chelating agents (e.g. EDTA) Storage in ethanol at low temperatures What are 5 popular sources of DNA Blood samples (must remain unclotted) collected in EDTA tubes Amniocytes (from amniocentesis fluid) tissue biopsies Cultured cells Paraffin-embedded tissue samples (mainly tumours) How are cells normally collected water mouth wash is used to dislodge epithelial cells lining the mouth Transfer your sample into the microcentrifuge tube and Centrifuge to extract cells During cell lysis, a lysis buffer is used. What does the buffer consist of? 50 mM Tris-HCI Buffer, (pH 8.0) + 1% SDS [sodium dodecyl sulfate] Topic 14 - Molecular Techniques 1  Tris buffer - maintain the pH of the solution for stable DNA 1% SDS - break nuclear membranes, allowing the DNA to be released into the solution 1% SDS - denature/unfolds proteins, making them more susceptible to protease cleavage How are proteins removed so DNA can be extracted (PPE) Phenolchloroformisoamyl alcohol extraction - removes debris from solution Protease Enzyme - destroy nuclear proteins that bind DNA + cytoplasmic enzymes that breakdown DNA, this increases the amount of intact DNA extracted. Ethanol - DNA precipitates Endonucleases… cut DNA into larger segments (i.e. Restriction Enzyme) Exonucleases…. cut DNA into very small fragments, nucleotides removed from ends They degrade DNA from the end of the molecule, they depolymerase it How do restriction enzymes in the bacteria not destroy bacterial DNA Their original function is to cleave bacteriophage DNA Bacterial DNA is methylated Topic 14 - Molecular Techniques 2  This prevent the enzyme from cleaving the host own DNA What is the role Alkaline Phosphatases during Modification of DNA Ends Alkaline Phosphatases – remove 5’ phosphate groups prevents re-joining of cut ends and self-ligation What is the role Terminal Transferases during Modification of DNA Ends transfer nucleotides to the 3’ end of a DNA molecule What is the role Nucleotide kinases during Modification of DNA Ends transfer single phosphate group from ATP to the 5’ end of DNA What is a vector Vehicle used to transfer genetic material to a target cell A DNA molecule capable of independent replication within a host organism Vector + Foreign DNA = Recombinant DNA Explain a A step-by-step example of how a restriction enzyme is used to add foreign DNA (eg. the human insulin gene) to a bacterial plasmid Cut Plasmid DNA + Target Gene with the same restriction enzyme restriction enzyme creates sticky ends that allow the foreign DNA and cloning vector (plasmid) to anneal ligase enzyme glues the annealed fragments together. ligated cloning vector is transformed into a bacterial host strain Topic 14 - Molecular Techniques 3  Explain how Drug Resistance Gene is transferred by Plasmids from cell to cell How does amplification + screening of a target gene occur Plating - Spread the transformed bacteria on a culture medium that includes the antibiotic resistance gene mentioned in the plasmid. Only bacteria with the plasmid (and thus antibiotic resistance gene) will survive. Plasmid Replication - Allow the bacteria to grow and replicate on the plate. The plasmid containing the target gene will also be replicated as the bacteria duplicate. Topic 14 - Molecular Techniques 4  As the bacteria grow, they replicate not only themselves but also the plasmid with gene. Population increases Selective Screening - identify colonies that likely contain the target gene. 2 Basic Properties of a Plasmid How does the LacZ Selection System Work? The LacZ gene, metabolizes X-gal, producing a blue product Topic 14 - Molecular Techniques 5  Insertion of foreign DNA into the MCS part of the LacZ gene destroys the LacZ gene, no blue product of X-gal formed in colonies (white) Topic 14 - Molecular Techniques 6  Why are the promoter regions of DNA so important? Determines the rate at which mRNA is synthesized. Amount of protein expressed depends on choice of promoter. What is microinjection Ensures entry and expression into the target cells. Require technical expertise and equipment, Only relatively few cells can be used Topic 14 - Molecular Techniques 7  What is Viral Transfection laboratory technique used to introduce genetic material into cells using viruses as carriers. process involves the use of viral vectors does not cause disease. what is the ‘bla’ Gene Bacteria with this gene grow in the presence of ampicillin what is the GFP gene Bacteria with this gene glow under near UV light Explain how Automated DNA Sequencing Occurs Automated DNA Sequencing: Laboratory technique to determine DNA nucleotide order. Primers attach DNA polymerase reads the template strand and synthesizes a new complementary second strand to match: strand will terminate synthesis when a “Terminator Base” attaches at the 3’ end Topic 14 - Molecular Techniques 8  normal bases have deoxyTTP (dTTP) but terminator bases have dideoxyTTP (ddTTP), which Lack 3'-OH group, stopping DNA strand elongation separate strands by Gel electrophoresis each terminator base makes up the sequence of DNA, reading from the bottom (5’ to 3’) Identifies the terminal nucleotide in each fragment. Sequence determined by analyzing fragment order (5’ to 3’) Topic 14 - Molecular Techniques 9  What are the 3 main variations of Protein Gel Electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Separates proteins based on size. Isoelectric focusing Separates proteins on the basis of charge - proteins will migrate up a pH gradient until they reach a pH equal to their pI (isoelectric point the pH at which a molecule carries no net electrical charge), where there will be no net charge and so the proteins will stop migrating. Two-dimensional electrophoresis (2D-PAGE) - size + charge Allows the separation of complex mixtures of proteins and is particularly important for diagnosing disease states in different tissues. what is the Human Genome Project (1986) to identify the approximate 100,000 (as believed at that time) genes in the human DNA. determine the sequences of the 3 billion bases that make up human DNA store this information in databases. develop tools for data analysis. Topic 14 - Molecular Techniques 10  What are some Ethical, legal and social implications of the Human Genome Project fairness in the use of genetic information privacy and confidentiality. psychological impact and stigmatization. genetic testing. reproductive issues. - education, standards, and quality control. Topic 14 - Molecular Techniques 11