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Gene Technology Continued Southern Blotting • Using an amplified gene as a probe to identify the same or a similar gene in another sample Degree of complementarity Varying conditions during hybridization will lead to: - High stringency – probe and fragment must be nearly identical - Low stringency – probe and fragment not as identical. Northern Blotting • Used to identify specific RNA molecules • Used to determine if transcription of a particular gene is transcribed in a particular cell type • Technique similar to Southern Blotting Western Blotting • Used to detect specific proteins • Use antibody as a probe (antibodies bind to specific sites on protein molecules) • Technique similar to Southern Blotting Determining the sequence of bases in a gene. DNA Sequencing • Dideoxy sequencing Synthesize deoxyribonucleotide without the -OH on the 3’ end called dideoxyribonucleotides Incorporation of dideoxyribonucleotides results in chain termination ddATP ddTTP ddGTP ddCTP Sequencing Movie • http://www.youtube.com/watch?v=UT9wqa VCH5s&feature=related Various uses • Within an organisms genome there are sections that are highly conserved – used to determine evolutionary relationships and there are sections that are highly variable – used to determine one individual from another • Restriction Fragment Length Polymorphism RFLP Other • Microsatellites – regions of repeat sequences e.g. AT rich region on Mitochondria – the repeat regions are highly variable and can be used to distinguish between sub populations. • DNA fingerprinting - makes use of microsatellites called minisatellites containing tandem repeats – also called ‘variable number of tandem repeats VNTR’s Biotech Uses • • • • Medicine Agriculture Biological Control Bioremediation How do we get genes into plants and animals? Gene transfer in plants Relatively easy. Many cells in plants are totipotent. Agrobacterium tumefaciens T DNA Tumour inducing Plasmid (Ti Plasmid) Adding genes to Animal DNA Mouse embryo stem cell Blastocyst Surrogate mother Young born will have recombinant DNA Selection Biotech Uses • Medicine - stem cell - insulin - gene therapy - genetic screening • Agriculture - herbicide resistant plants - resistant to freezing, shipping • Biological Control - cotton and BT gene • Bioremediation - bacteria engineered to eat toxic substances
