Upload
aubrie-lane
View
214
Download
0
Embed Size (px)
Citation preview
Gene TechnologyContinued
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 sequencingSynthesize deoxyribonucleotide without the -OH on the 3’ end called dideoxyribonucleotidesIncorporation of dideoxyribonucleotides results in chain
termination
ddATPddTTPddGTPddCTP
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 inducingPlasmid (Ti Plasmid)
Adding genes to Animal DNAMouse 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