DNA Technology Lecture Notes (BO101 2024)

DNA Technology BO101 - Molecular Genetics - Lecture 6 Dr Andrew Flaus, Biochemistry What is DNA technology ๏ DNA technology ‣ Manipulation of genes and organisms for practical purposes ๏ Traditional ‣ Breeding of plants and animals ‣ Use of microorganisms to make bread, wine, beer, ch...

DNA Technology BO101 - Molecular Genetics - Lecture 6 Dr Andrew Flaus, Biochemistry What is DNA technology ๏ DNA technology ‣ Manipulation of genes and organisms for practical purposes ๏ Traditional ‣ Breeding of plants and animals ‣ Use of microorganisms to make bread, wine, beer, cheese ๏ Biotech revolution ‣ Genetic engineering ‣ Therapeutic cloning Campbell extra images Genes are portable ๏ DNA cloning ‣ Preparing multiple identical copies of a gene or DNA segment ‣ Genetic engineering ๏ Reasons ‣ Universality of molecular genetics ‣ Genes can be moved around and manipulated ‣ Applications in diverse areas of biotechnology Campbell g 19.4 fi Recombinant DNA engineering ๏ Recombinant DNA ‣ DNA assembled in an order not found in nature ๏ Plasmid ‣ Small circle of DNA that can be retained by bacterial cells ๏ Restriction enzyme ‣ Enzyme that recognises and cuts speci c DNA sequence sites Campbell g 19.5 fi fi Simple cut-paste steps for “cloning” 1. Cut DNA with restriction enzyme ‣ Enzyme recognises DNA sequence and cuts DNA helix 2. Mix DNA fragments ‣ Compatible sticky ends 3. Paste with DNA ligase enzyme ‣ Forms bonds to make continuous DNA helix again Campbell g 19.5 fi Isolating a unique clone ๏ Simple cut-paste steps ๏ Transformation ‣ Induce bacteria to take up plasmid ๏ Selection ‣ Plasmid also contains an antibiotic resistance gene ‣ Only bacteria with plasmid survive ๏ Clone ‣ All descendent bacteria now carry recombinant DNA in plasmid Campbell g 19.8 fi PCR enables ampli cation of DNA copies fi  PCR for DNA copies ๏ PCR ‣ Polymerase Chain Reaction ๏ Ampli cation ‣ Doubles each cycle Can make huge numbers of copies ‣ Can amplify from a complex mixture of DNA Useful for cloning, diagnostics ๏ Speci city ‣ Small synthetic DNA ‘primers’ de ne ends of target to amplify Campbell g 19.7 fi fi fi fi  PCR cycle steps 1. Denaturation ‣ Separate DNA strands by heating 2. Annealing ‣ All a speci c pair of primers to bind to single strands ‣ De nes ends of region to amplify 3. Extension ‣ Allow DNA polymerase to extend from primers ‣ Doubles copies of region between primers in each cycle Campbell g 19.7 fi fi fi Applications of DNA technology Forensic applications ๏ Polymorphisms ‣ Unique differences at sites on chromosomes between individuals (eg STRs) ๏ Example: DNA forensics ‣ Earl Washington convicted to rape and murder in 1992 ‣ DNA analysis of Short Tandem Repeat (STR) copies in 2000 ‣ Released from prison 2001 ‣ Actual murderer found, convicted Campbell g 19.24 fi Biopharmaceutical applications ๏ Strategy 1. Amplify human gene for useful protein by PCR 2. Clone gene into plasmid 3. Express and purify protein ๏ Example products ‣ Insulin to treat diabetes ‣ Antibodies to treat cancer ‣ Growth hormone for dwar sm ‣ Tissue plasminogen activator to treat heart attacks Campbell g 19.4 fi fi Agricultural applications ๏ ‘Pharm’ animals ‣ Genome engineer animals to secrete useful molecules Anti-coagulents in goat milk ‣ Compete with cell-based systems ๏ Transgenic crops ‣ Genome engineer plants with desirable traits ‣ Prevalent in some parts of world but not accepted in all (Europe) Pest- and herbicide-resistance Improved nutrition Campbell g 35.25 fi Regenerative medicine Plant organismal cloning ๏ Entire plants can be cloned from single cells ‣ A mature single plant cell can “dedifferentiate” to be totipotent ‣ Totipotent cells can be grown to give all cells of mature plant ๏ Totipotency ‣ Capability of a cell to give rise to any cell type Campbell ed10 g 19.15 fi Animal organismal cloning ๏ Totipotency ‣ Capability of a cell to give rise to any cell type ‣ Totipotency is more dif cult to achieve in animal cells ๏ Organismal cloning ‣ Create whole organisms genetically identical to single cell ‣ Requires totipotent cell ‣ Possible in animals by nuclear transplantation Example: Dolly the sheep Campbell ed10 g 19.19 fi fi Dolly the Sheep, 1997 ๏ Organismal cloning ๏ Nuclear transplantation ‣ Egg cell with nucleus removed ‣ Donor nucleus from an adult cell ๏ Problems ‣ Low ef ciency (