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 VCH5s&feature=relatedhttp:// 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