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LECTURE 16 – Using Genomic Variation for Identity DNA Level Variations • Inherited as any other locus and they are Co-dominant • These markers can be used to determine which parent the allele came from • Individuals can be identified by their unique DNA profile DNA Manipulation • We may want to identify the function of a gene by: Ø Determining when the gene is expressed Ø Loss of gene function by deleting the gene to see the affects Ø Addition of genes How is DNA Manipulated 1. Identify the Gene/DNA of interest 2. Isolate the gene into a vector (cloning) Ø Generally, plasmids are used as a vector 3. Make necessary changes to the DNA – join the DNA fragments 4. Reintroduce the construct into our organism of choice • Genetics Toolbox: Ø PCR – amplify many copies of specific region of DNA Ø Plasmids – vectors that carry genes in bacteria, they are self replicating circles of DNA Ø Antibiotic resistance – select for a recombinant DNA molecule Ø Restriction enzymes – cut DNA molecules at specific sequence Ø DNA ligase – join DNA fragments together Ø Transformation – uptake of exogenous DNA Ø Gel electrophoresis – separate fragments on the basis of size Ø Hybridisation – identify location of specific nucleotide sequence Ø Sequencing – identify sequence of molecule of DNA • Restriction Endonucleases/Enzymes Ø Bacteria can be infected by viruses known as bacteriophage Ø Inbuilt immunity in the bacteria against bacteriophage is the restriction enzyme – cleaves the DNA of the bacteriophage Ø Bacteria protects its own DNA from the restriction enzymes (by methylation) Ø Restriction enzymes cut the DNA leaving a sticky end (overhang of one DNA strand) or a blunt end (strands cut at same point) Ø Restriction enzymes will only cut certain sequences of bases in the DNA • DNA ligase Ø Joins the DNA fragments together in vitro – complementary ends Ø Ligation can occur to allow the gene of interest into the vector but it can also join the DNA of the plasmid back together again (empty vector) • Transformation Ø Heat shock the bacteria which means it will take up the vector Ø Transformed bacteria can grow as it has antibiotic resistance due to taking up the plasmid – thereby selected by growing on plate with antibiotic