CULTURE INDEPENDENT ANALYSIS OF MICROBIAL COMMUNITIES IN SOIL

1. CULTURE INDEPENDENT ANALYSIS OF MICROBIAL COMMUNITIES IN SOIL Molecular Microbial Ecology

2. Culture-Independent Soil Analysis • Isolate DNA from soil • PCR amplify 16S rRNA genes • Agarose gel electrophoresis • Clone into vector • Transform into E. coli and selection on media • Plasmid preps • Restriction enzyme digest • Dideoxy sequencing • Sequence analysis

3. DNA Purification

4. Polymerase Chain Reaction • Used to amplify DNA in a sample • Specific primers are used to amplify a specific region of DNA – not all of it • Primers are synthetically produced to be complementary to the region of interest • Taq polymerase – DNA polymerase • Enzyme that synthesizes new DNA strands • Obtained from Thermusaqauticus • Thermophile isolated from Yellowstone • Works at 72°C

5. Melting and Annealing DNA • Melting or denaturation • Renaturation or annealing

6. PCR • Repeated cycles of DNA synthesis • Denaturation • Heat to 94°C • Separate DNA strands from each other • Annealing (Priming) • Cool to 50° to 65°C • Temperature depends on Tm of primers • Primers hybridize to DNA template • Extension (Synthesis) • Heat to 72°C • DNA synthesis • Repeat the above 3 steps 20 to 40 X

7. PCR • http://www.dnalc.org/resources/3d/19-polymerase-chain-reaction.html • http://www.dnalc.org/resources/animations/pcr.html

8. Designing Thermal Cycler Protocols

9. Troubleshooting PCR • Annealing temperature • If not enough product, lower temp • If too much background smearing, increase temp • Touchdown annealing cycles • Increase MgCl2 concentration • Increased salts increase Tm of primer • Adjust primer concentrations

10. Agarose Gel Electrophoresis • Used to separate DNA fragments from each other • Gel is a matrix of agarose • Crosslinks with pores through which molecules can move • Electrical field is passed through the agarose gel • DNA is negatively charged and will move towards the positive pole of the gel • Smaller molecules will move faster

11. Gel Electrophoresis To visualize the bands it is stained with a fluorescent dyewhich fluoresces under UV light or with methylene blue Figure 10.4

12. Loading Gel

13. Running Gel

15. Topo TA Cloning

16. Topo TA Cloning • Selection can be on LB + kanamycin or ampicillin • Ampicillin can lead to satellite colonies • If no insert, vector should not be able to close up • If it does circularize, enzyme is produced that kills cells

17. Restriction Endonucleases • Cleave double stranded DNA at specific sequences • Named for the bacteria from which they are isolated

19. DNA Sequencing • Technique used to determine exact order of nucleotides (bases) of DNA • Uses dideoxynucleotides (ddNTPs) • Lacking the 3’ hydoxyl group on the sugar • When a ddNTP is incorporated into a growing DNA strand, elongation terminates

20. DNA Sequencing

22. http://www.dnalc.org/resources/animations/cycseq.html