Download LECTURE 16 – Using Genomic Variation for Identity DNA Level

LECTURE 16 – Using Genomic Variation for Identity
DNA Level Variations
• Inherited as any other locus and they are Co-dominant
• These markers can be used to determine which parent the allele came from
• Individuals can be identified by their unique DNA profile
DNA Manipulation
• We may want to identify the function of a gene by:
Ø Determining when the gene is expressed
Ø Loss of gene function by deleting the gene to see the affects
Ø Addition of genes
How is DNA Manipulated
1. Identify the Gene/DNA of interest
2. Isolate the gene into a vector (cloning)
Ø Generally, plasmids are used as a vector
3. Make necessary changes to the DNA – join the DNA fragments
4. Reintroduce the construct into our organism of choice
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Genetics Toolbox:
Ø PCR – amplify many copies of specific region of DNA
Ø Plasmids – vectors that carry genes in bacteria, they are self replicating circles of
DNA
Ø Antibiotic resistance – select for a recombinant DNA molecule
Ø Restriction enzymes – cut DNA molecules at specific sequence
Ø DNA ligase – join DNA fragments together
Ø Transformation – uptake of exogenous DNA
Ø Gel electrophoresis – separate fragments on the basis of size
Ø Hybridisation – identify location of specific nucleotide sequence
Ø Sequencing – identify sequence of molecule of DNA
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Restriction Endonucleases/Enzymes
Ø Bacteria can be infected by viruses known as bacteriophage
Ø Inbuilt immunity in the bacteria against bacteriophage is the restriction
enzyme – cleaves the DNA of the bacteriophage
Ø Bacteria protects its own DNA from the restriction enzymes (by methylation)
Ø Restriction enzymes cut the DNA leaving a sticky end (overhang of one DNA
strand) or a blunt end (strands cut at same point)
Ø Restriction enzymes will only cut certain sequences of bases in the DNA
•
DNA ligase
Ø Joins the DNA fragments together in vitro – complementary ends
Ø Ligation can occur to allow the gene of interest into the vector but it can also
join the DNA of the plasmid back together again (empty vector)
•
Transformation
Ø Heat shock the bacteria which means it will take up the vector
Ø Transformed bacteria can grow as it has antibiotic resistance due to taking up
the plasmid – thereby selected by growing on plate with antibiotic