CULTURE INDEPENDENT ANALYSIS OF MICROBIAL COMMUNITIES IN SOIL Molecular Microbial Ecology

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

DNA Purification

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 Thermus aqauticus Thermophile isolated from Yellowstone Works at 72°C

Melting and Annealing DNA Melting or denaturation Renaturation or annealing

PCR Repeated cycles of DNA synthesis Denaturation Annealing (Priming) 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

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

Designing Thermal Cycler Protocols

Troubleshooting PCR Annealing temperature Touchdown annealing cycles 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

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

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

Loading Gel

Running Gel

Topo TA Cloning

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

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

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

DNA Sequencing

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

MNF001 and MNF003 Culture Independent Analysis