Molecular Methods in Microbial Ecology

1. Molecular Methods in Microbial Ecology Contact Info: Julie Huber Lillie 305 x7291 jhuber@mbl.edu Schedule: 21 October: Introductory Lecture, DNA extraction 23 October: Run DNA products on gel Lecture on PCR Prepare PCR reactions 28 October: 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

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

4. The Solution: Molecular Biology DNA Transcription mRNA Translation Ribosome Protein • Are present in all cells- Bacteria, Archaea and Eukaryotes • Are documents of evolutionary history • Are the basis of all molecular biological techniques

6. Head et al. 1998

7. Head et al. 1998

8. DNA extraction from Winogradsky Columns

9. 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

10. 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

12. Head et al. 1998

13. Head et al. 1998

14. 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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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. DNA Transcription mRNA Translation Ribosome Protein Molecular Biology • Are present in all cells- Bacteria, Archaea and Eukaryotes • Are documents of evolutionary history • Are the basis of all molecular biological techniques

18. 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

19. 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

20. SSU rRNA

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

22. 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

23. 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

24. Typical PCR Profile

25. Slide courtesy of Byron Crump

26. 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…

27. Primers we are using • 16S Bacteria • 16S Archaea • mcr Methanogens (Methyl coenzyme M reductase) • dsr Sulfate reducers (Dissimilatory bisulfite reductase

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

30. Class DNA 10 kb Andrea Paliza Jessica Sarah Rob Amy

31. Rob Sarah Paliza Jessica Andrea Amy

32. Rob Sarah Paliza Jessica Andrea Amy

33. 3 kb 1 kb 3 kb 1 kb 1 1 1 1 1 1 2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5 5 5 5 6 6 6 6 6 6 Rob Andrea Paliza Sarah Amy Jessica

34. So you have a positive PCR product: Now what? • Clone and sequence clones • Go straight into sequencing (454) • Get “community fingerprint” via DGGE and sequence bands • Get “community fingerprint” via T-RFLP

35. Schematic courtesy of B. Crump

39. The 454 Approach Schematic courtesy of B. Crump

40. The 454 Approach 400,000 reads 250 bp in length Average ~ 250 bp

41. 454 • Avoids laborious/expensive cloning procedures • Sample diversity deeply and quickly • Find “rare” or low abundance organisms • Limited to short reads (<250bp) • 454 has revolutionized sequencing- many new technologies every day!

43. What if we wanted to compare the general community composition of each column rather than getting detailed taxonomic information? FINGERPRINTING METHODS

44. Schematic courtesy of B. Crump

46. Schematic courtesy of B. Crump

48. Schematic courtesy of B. Crump