Transcript Gene Technology Continued

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
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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