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