Galkiewicz JP, Kellogg CA 2008 Cross-kingdom amplification using Bacteria-specific primers: Complications for studies of

1. Galkiewicz JP, Kellogg CA (2008) Cross-kingdom amplification using Bacteria-specific primers: Complications for studies of coral microbial ecology. Appl Environ Microbiol 74:7828-7831

2. Coral Microbial Ecology Studies interactions between corals and associated microbes Associated microbes include algae, bacteria, fungi and archaea Functional roles of these microbes are not well explored

3. Microbial Identification Both culture-based & culture-independent methods used Marine bacteria present culturing difficulties, so culture-independent methods have predominated

4. Culture-Independent Identification PCR amplification of target DNA sequence 16S rRNA gene is most commonly used for bacterial identification PCR Video

5. Study Goals Show that non-specific amplification using these common primers (8F/1492R) can occur. Propose an alternative method that addresses this issue.

6. Sample Collection Collected Pocillopora damicornis 3 locations 2 American Samoa 1 NW Hawaiian Islands 2 samples per location Preserved in DMSO-EDTA-salt buffer

7. DNA Extraction DNA extracted using Mo Bio PowerSoil kit How was tissue collected & separated from skeleton?

8. PCR Master Mix: 12.5 �L AmpliTaq Gold 10 pM each primer 9.5 �L sterile deionized water 1 �L DNA template Conditions: initial 15 minutes @ 95�C 30 cycles: 1 minute @ 95�C 1 minute @ 54�C 2 minutes @ 72�C final 10 minutes @ 72�C

9. Cloning All six samples were amplified & cloned separately Only first library was sequenced Purified initial PCR products & cloned them using Qiagen kits

10. Sequencing PCR of cloned vectors with M13/pUC primers (unknown conditions) Screened using 1% agarose gel to select appropriately-sized amplicons Sequenced only in forward direction

11. Sequence Analysis Raw sequences cleaned-up using phred & Greengenes software BLAST searches to find closest matches E-value = 0 similarity >96% indentity >95%

12. Results 100 of 101 sequences were most similar to a coral 18S rRNA gene 1 sequence was most similar to the endosymbiotic dinoflagellate, Symbiodinium sp., found in coral tissue

13. Problem Enough similarity between coral 18S rRNA gene sequences and �bacteria specific� primers to amplify Coral amplicon rough same length as bacterial amplicon ? Cannot differentiate bacterial & coral amplicons on a gel

14. Solution Use alternate primer set (63F/1542R) Much lower homology between primers & coral 18S rRNA gene sequences ? Less likely to anneal or amplify coral sequences If amplified, coral amplicon is significantly shorter than bacterial amplicons ? Can isolate bacterial amplicons on a gel

15. Differentiation of PCR products Lanes 1-3: alternative primer pair (63F/1542R) 1 = negative control 2 = bacterial DNA (positive control) 3 = sample Lanes 1-3: current primer pair (8F/1492R) 4 = coral tissue 5 = bacterial DNA (positive control) 6 = negative control

16. Importance of Annealing Temperature 63F primer may show amplification bias Altering the annealing temperature may address this lower TA = lower specificity High temperatures used in previous studies probably prevented non-bacterial amplification May have missed some important bacteria

17. Final Thoughts Similar results have been found in other systems, e.g., wine Primer sets should be periodically reassessed as number of published sequences increases Tune specificity of primers using conditions or degenerate primers to optimize bacterial identification Important to understanding functional roles