1. MOLECULAR GENETICS TECHNIQUES

2. Molecular Genetics Technologies i.Polymerase chain reaction ii.DNA/Genomic sequencing iii.Gel electrophoresis iv.Restriction enzymes v.Plasmids/recombinant DNA vi.Transformations

3. A real life genetics project… (don’t take this note) We want to decontaminate crop soil that has been sprayed with pesticide We found a bacteria in Yellowstone National Park that eats pesticides We want to put the enzyme responsible into something commonly found all over the world (e.g. Pseudomonas)

4. Polymerase chain reaction (PCR) Uses repeating cycles of heating/cooling to amplify target gene(s) Primers are used to identify the region to be copied Makes many (10 4 - 10 6 ) copies Resulting gene copies can be used in other techniques

7. DNA Sequencing Used to read the order of base pairs within a genome Old: Sanger method New: ‘next generation’ Massive, worldwide database of all known sequences continues to grow…

9. Agarose gel electrophoresis Allows us to separate DNA fragments based on length We load a DNA sample (eg. from a PCR) into slots in an agarose gel Agarose is a meshwork of cross- linked molecules through which DNA has trouble moving An electric field is applied to the apparatus

11. Agarose gel electrophoresis DNA is negatively charged and will migrate through the gel towards the anode (+) DNA separates based on length Longer DNA fragments move slowly Shorter DNA fragments move quickly Visualize DNA using UV light and can cut out and purify DNA of choice

12. Restriction enzymes Restriction enzymes cut our bacteria at known sequences Cutting DNA in this way allows us to transplant genes in meaningful ways The specific cut site is called a restriction site and may be left with ‘sticky’ or ‘blunt’ ends

13. Restriction enzymes By incubating our DNA with restriction enzymes we ‘digest’ it We can rescue the fragments of desired length by doing agarose gel electrophoresis Fragments can be used to make recombinant DNA…

14. Activity

15. Plasmids/Recombinant DNA In addition to their genomic DNA, bacteria can possess plasmids Plasmids contain one or a few genes that can be passed among bacteria We can engineer a plasmid to contain our gene and put it inside bacteria to transcribe/translate the protein

16. Plasmids/Recombinant DNA

17. Transformations The process of getting recombinant DNA into bacteria is called transformation Transformations can be done using electricity or temperature shocks to entice bacteria to take up the new plasmid http://labcenter.dnalc.org/labs/transformation/transformation_h.html