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

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5. HabitatCulturability (%) Seawater 0.001-0.1 Freshwater 0.25 Sediments 0.25 Soil 0.3 From Amann et al. 1995 Microbiological Reviews The Challenge for Microbial Ecology How do you study something you can’t grow in the lab?

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

8. Head et al. 1998

10. DNA extraction from Winogradsky Columns

11. DNA Extraction 1.Lyse cell membrane a.Chemically  detergent b.Physically  bead beating 2.Pellet cell membrane, proteins and other cell parts while DNA stays in solution 3.Remove other inhibitors from DNA 4.Mix DNA with acid and salt  stick to filter 5.Wash filter-bound DNA several times with alcohol 6.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. L RB MC GG AS BP LS Genomic DNA The sum total of all DNA from an organism or a community of organisms

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

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

22. SSU rRNA

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

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, MgCl 2, etc) A set of primers, one Forward, one Reverse Template DNA

26. Typical PCR Profile TemperatureTimeAction 95ºC5 minutesDNA Taq polymerase activation 35 cycles of: 95ºC 54ºC 72ºC 1 minute DNA denaturization Primer annealing Extension creation 72ºC10 minutesFinal extension created

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 MgCl 2 Concentration of template DNA Number of replication cycles Etc…

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

30. Luton et al. 2002 16S rDNAmcrA

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 10 kb 3 kb 500 bp Carrie Chrissy Amy Haruka

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. Carrie Marshall ChrissyKenly Amy NobuHaruka Monica 3 kb 500 bp 3 4 2 1 3 kb 500 bp

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

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

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

48. BLAST Basic 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

51. Classification (who is who)

52. Luton et al. 2002 16S rDNAmcrA

54. B. Crump

57. Built clone libraries from deep-sea rocks Compared them to one another and other habitats

58. Santelli et al. 2008

60. Community Overlap Santelli et al. 2008

61. 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)

62. Schematic courtesy of B. Crump MPS Approaches

63. From Hugenholtz and Tyson 2008 PlatformMillion base pairs per run Cost per base (cents) Average read length (base pairs) Dye-terminator (ABI 3730xl) 0.070.1700 454-Roche pyrosequencing (GSFLX titanium) 4000.003400 Illumina sequencing (GAii) 2,0000.000735

65. 3,000 species? How many species in 1 L of vent fluid?

66. 3,000 species? > 36,000 species! How many species in 1 L of vent fluid?

67. Now we know who is there: What next? Quantify particular groups: FISH or qPCR

68. Head et al. 1998

69. Fluorescent In-Situ Hybridization (FISH) B. Crump

70. Fluorescent In-Situ Hybridization (FISH) Schleper et al. 2005

71. Quantitative (Real Time) PCR Real time PCR monitors the fluorescence emitted during the reactions as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection

72. Detection of “amplification-associated fluorescence” at each cycle during PCR No gel-based analysis Computer-based analysis Compare to internal standards Must insure specific binding of probes/dye Quantitative (Real Time) PCR

73. Quantitative PCR

74. Now we know who and how many: What next? Metagenomics RNA-based methods Many many more…

75. Metagenomics a.k.a., Community Genomics, Environmental Genomics Does not rely on Primers or Probes (apriori knowledge)! Image courtesy of John Heidelberg

76. Metagenomics

78. Access genomes of uncultured microbes: Functional Potential Metabolic Pathways Horizontal Gene Transfer … Metagenomics

79. From the Most “ Simple ” Microbial Communities … Acid Mine Drainage (pH ~0!) Jillian Banfield (UC Berkeley) Well-studied, defined environment with ~4 dominant members Were able to reconstruct almost entire community “metagenome” Tyson et al. 2004

80. … to the potentially most diverse! The Sorcerer II Global Ocean Sampling Expedition J. Craig Venter Institute “Sequence now, ask questions later” Very few genomes reconstructed Sequenced 6.3 billion DNA base pairs (Human genome is ~3.2) from top 5 m of ocean Discovered more than 6 million genes… and they are only halfway done! Venter et al. 2004

81. Most of these methods are “who is there” not “who is active” Use RNA Link FISH with activity/uptake DNA mRNA Transcription Protein Translation Ribosome

82. Reverse Transcription PCR (RT-PCR) Looks at what genes are being expressed in the environment Isolate mRNA Reverse transcribe mRNA to produce complementary DNA (cDNA) Amplify cDNA by PCR Analyze genes from environment

83. RT-PCR RNA + Reverse Transcriptase + dNTPs= cDNA cDNA + Primers + Taq + dNTPs = gene of interest Who is active? What genes are active?

84. Metatranscriptomics Access expressed genes of uncultured microbes

85. (Some) Problems with Molecular Methods D/RNA extractionIncomplete sampling Resistance to cell lysis StorageEnzymatic degradation PCRInhibitors in template DNA Amplification bias Gene copy number Fidelity of PCR Differential denaturation efficiency Chimeric PCR products AnytimeContamination w/ non-target DNA

86. The “best approach?” A little bit of everything!

87. And the list goes on… Optical tweezers Single cell genomics Meta-proteomics Microarrays Flow Cytometry Nano-SIMS FISH In-situ PCR and FISH …