Gene Technology Continued. Southern Blotting Using an amplified gene as a probe to identify the same...
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Transcript of Gene Technology Continued. Southern Blotting Using an amplified gene as a probe to identify the same...
![Page 1: Gene Technology Continued. Southern Blotting Using an amplified gene as a probe to identify the same or a similar gene in another sample.](https://reader036.fdocuments.us/reader036/viewer/2022070610/5a4d1bb37f8b9ab0599cd72b/html5/thumbnails/1.jpg)
Gene TechnologyContinued
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Southern Blotting
• Using an amplified gene as a probe to identify the same or a similar gene in another sample
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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.
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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
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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
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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
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Sequencing Movie• http://www.youtube.com/watch?v=UT9wqa
VCH5s&feature=related
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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
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• Restriction Fragment Length Polymorphism RFLP
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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
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Biotech Uses
• Medicine• Agriculture• Biological Control• Bioremediation
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How do we get genes into plants and animals?
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Gene transfer in plants
Relatively easy.
Many cells in plants are totipotent.
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Agrobacterium tumefaciens
T DNA Tumour inducingPlasmid (Ti Plasmid)
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Adding genes to Animal DNAMouse embryo stem cell
Blastocyst
Surrogate mother
Young born will have recombinant DNA
Selection
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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