Molecular Methods in Microbial Ecology

1. Molecular Methods in Microbial Ecology Contact Info: Julie Huber Lillie 305 x7291 jhuber@mbl.edu Schedule: 22 Sept: Introductory Lecture, DNA extraction 24 Sept: Run DNA products on gel Lecture on PCR Prepare PCR reactions 29 Sept: Analyze gels from PCR Lecture on other molecular methods Readings: Head et al. 1998. Microbial Ecology 35: 1-21.

2. Introduction to molecular methods in microbial ecology Extract DNA from Winogradsky Columns Day 1

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5. The Challenge for Microbial Ecology How do you study something you can’t grow in the lab? From Amann et al. 1995 Microbiological Reviews

6. The Solution: Molecular Biology DNA Transcription mRNA Translation Ribosome Protein • Present in all cells- Bacteria, Archaea and Eukaryotes • Documents of evolutionary history • Basis of all molecular biological techniques

8. Head et al. 1998

9. Head et al. 1998

10. DNA extraction from Winogradsky Columns

11. DNA Extraction • Lyse cell membrane • Chemically  detergent • Physically  bead beating • Pellet cell membrane, proteins and other cell parts while DNA stays in solution • Remove other inhibitors from DNA • Mix DNA with acid and salt  stick to filter • Wash filter-bound DNA several times with alcohol • Elute DNA off membrane with pH 8, low-salt buffer

12. Day 2 • Run an electrophoresis gel of the DNA products extracted from your columns • Learn about PCR • Set up PCR reactions using the DNA from your extractions and an assortment of primers

13. Basics of Gel Electrophoresis • The gel is a matrix (like jello with holes) • DNA is negatively charged- will run to positive • Smaller fragments run faster than larger ones • Gel contains Ethidium Bromide, which binds to DNA and fluoresces when hit with UV light (WEAR GLOVES!!!)

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15. Genomic DNA The sum total of all DNA from an organism or a community of organisms L RB MC GG AS BP LS

16. What to do • Mix 10 µl of your DNA with 2 µl loading buffer • Load in well on gel • I’ll load the ladder • Run it • Take a picture of it

17. Head et al. 1998

18. Head et al. 1998

20. The Star of the Show: SSU rRNA • Everybody has it • Contains both highly conserved and variable regions • -allows making comparisons between different organisms • over long periods of time (evolutionary history) • Not laterally transferred between organisms • HUGE and growing database

21. 21 different proteins 16S rRNA 30S subunit 31 different proteins 70S Ribosome 50S subunit 5S rRNA 23S rRNA Ribosomes • Make proteins • rRNA is transcribed from rDNA genes

22. SSU rRNA

23. BACTERIA Universal Tree of Life BACTERIA ARCHAEA ARCHAEA You Are Here EUKARYA EUKARYA Modified from Norman Pace

24. Polymerase Chain Reaction (PCR) • Rapid, inexpensive and simple way of making millions of copies of a gene starting with very few copies • Does not require the use of isotopes or toxic chemicals • It involves preparing the sample DNA and a master mix with primers, followed by detecting reaction products

25. Every PCR contains: • A DNA Polymerase (most common, Taq) • Deoxynucleotide Triphosphates (A, C, T, G) • Buffer (salt, MgCl2, etc) • A set of primers, one Forward, one Reverse • Template DNA

26. Typical PCR Profile

27. Slide courtesy of Byron Crump

28. Things you can optimize • Temperature and time to activate Taq polymerase • Temperature and time to allow primer annealing • Temperature and time for extension • Concentration of reagents, especially primers, dNTPs, and MgCl2 • Concentration of template DNA • Number of replication cycles • Etc…

29. Beyond 16S • Identical 16S = Identical Function • Target functional genes

30. 16S rDNA mcrA Luton et al. 2002

31. Primers we are using • 16S rRNA Bacteria • 16S rRNA Archaea • mcrA Methanogens • Methyl coenzyme M reductase • dsrB Sulfate reducers • Dissimilatory bisulfite reductase

33. Day 3 • Examine gels from DNA and PCR • Learn about more molecular methods in microbial ecology

34. Class DNA Nobu Monica Kenly Marshall Carrie Chrissy Amy Haruka 10 kb 3 kb 500 bp

37. Some Problems with PCR • Inhibitors in template DNA • Amplification bias • Gene copy number • Limited by primer design • Differential denaturation efficiency • Chimeric PCR products may form • Contamination w/ non-target DNA • Potentially low sensitivity and resolution • General screw-ups

38. Amy Nobu Haruka Monica 3 kb 500 bp 3 4 2 1 3 4 2 1 3 4 2 1 3 4 2 1 Carrie Marshall Chrissy Kenly 3 kb 500 bp 3 4 2 1 3 4 2 1 3 4 2 1 3 4 2 1

39. So you have a positive PCR product: Now what? • Get “community fingerprint” via T-RFLP • Get “community fingerprint” via DGGE and sequence bands • Clone and sequence clones • Go straight into sequencing (massively parallel sequencing, MPS)

40. B. Crump

43. B. Crump

45. B. Crump

46. What do you DO with sequences? • Perform a similarity search (database) • Align the sequences (common ancestry) • Build a tree (phylogeny and taxonomy)

47. BLASTBasic Local Alignment Search Tool http://blast.ncbi.nlm.nih.gov/Blast.cgi

48. BLASTBasic Local Alignment Search Tool http://blast.ncbi.nlm.nih.gov/Blast.cgi

49. Align Sequences and Relatives

50. Build a Tree (Phylogeny) Reconstructing evolutionary history and studying the patterns of relationships among organisms