1. Molecular Methods in Microbial Ecology
Contact Info: Julie Huber
Lillie 305
x7291
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 Microbial Ecology 35: 1-21.

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

3. The Challenge for Microbial Ecology
Habitat
Culturability (%)
Seawater
Freshwater
0.25
Sediments
Soil
0.3
How do you study something you can’t grow in the lab?
From Amann et al 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 7

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 12

13. Head et al. 1998 13

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!!!)

15. - - - - - - - - - - -

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

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

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. Universal Tree of Life BACTERIA BACTERIA ARCHAEA ARCHAEA EUKARYA
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 Temperature Time Action 95ºC 5 minutes
DNA Taq polymerase activation
35 cycles of: 95ºC 54ºC 72ºC
1 minute
DNA denaturization
Primer annealing
Extension creation
72ºC
10 minutes
Final extension created

25. Slide courtesy of Byron Crump
PCR. This graphic is meant to explain how PCR amplifies or makes many copies of a target gene. Here is the target gene on a long strand of genomic DNA. Short sequences of 10 to 30 bases on both ends of the gene, called primer sites, are figured out in advance and are used to direct the reaction to that gene. The reaction begins when the double stranded DNA is denatured into two single strands. Those short pieces of single-stranded DNA called primers match up with the DNA sequences of the primer sites and bind to them. An enzyme called DNA polymerase then takes free nucleotides that are included in the reaction mixture and builds complimentary strands of DNA starting at the primers. Now we have two copies of the original gene. The steps are repeated and we have 4 copies. So after 30 cycles of PCR there is a tremendous number of copies of the target gene.
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. Rob Andrea Paliza Sarah Amy Jessica 1 4 3 2 5 6 1 4 3 2 5 6 1 4 3 2 5
3 kb
1 kb
3 kb
1 kb 1 4 3 2 5 6 1 4 3 2 5 6
Rob
Andrea 1 4 3 2 5 6 1 4 3 2 5 6
Paliza
Sarah 1 4 3 2 5 6 1 4 3 2 5 6
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
Clone Library sequencing: The first method is the method used in some of the original papers on the diversity of natural bacterial communities, and it is called clone library sequencing. DNA is extracted from environmental samples, including DNA from bacteria, flagellates, diatom chains, everything. PCR is used with primers designed to amplify 16S rRNA genes from all the bacteria in the sample. The rest of the method separates these genes so they can be analyzed individually. The 16S rRNA genes are put into circular DNA called plasmids and inserted into E. coli cells such that there is one plasmid per cell. The E. coli cells are then grown up on plates such that each colony contains many copies of one of these 16S rRNA genes. The plasmids are then extracted from these colonies and sequenced.
Schematic courtesy of B. Crump

39. The 454 Approach Schematic courtesy of B. Crump
Clone Library sequencing: The first method is the method used in some of the original papers on the diversity of natural bacterial communities, and it is called clone library sequencing. DNA is extracted from environmental samples, including DNA from bacteria, flagellates, diatom chains, everything. PCR is used with primers designed to amplify 16S rRNA genes from all the bacteria in the sample. The rest of the method separates these genes so they can be analyzed individually. The 16S rRNA genes are put into circular DNA called plasmids and inserted into E. coli cells such that there is one plasmid per cell. The E. coli cells are then grown up on plates such that each colony contains many copies of one of these 16S rRNA genes. The plasmids are then extracted from these colonies and sequenced.
Schematic courtesy of B. Crump

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

41. 454 Sample diversity deeply and quickly
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. FINGERPRINTING METHODS
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
DGGE: The method I have been working with is called denaturing gradient gel electrophoresis. 16S rRNA genes are amplified using PCR just like the clone libraries. These pieces of DNA have different sequences because they are from different organisms, and the differences in the sequences means that they will denature from double-stranded to single-stranded DNA at different concentrations of chemical denaturant. So the mixture of different genes are run on an acrylamide gel containing a gradient of denaturing chemicals, and the different genes denature and stop moving down the gel when they reach their critical denaturant concentration. The result is that each band on the gel represents a different organism or group of organisms from your original sample.
Schematic courtesy of B. Crump

46. Schematic courtesy of B. Crump

48. Schematic courtesy of B. Crump
TRFLP: The first one is called Terminal Restriction Fragment Length Polymorphism or TRFLP. The first part of the method is the same as Clone Library sequencing, but one of the primers is labeled with a fluorescent molecule or with P-32. The mix of PCR products are then cut up with a restriction enzyme. Restriction enzymes seek out very specific short sequences on a piece of DNA and they cut the DNA only at those sequences. In this example I am using a restriction enzyme that cuts at the sequence “ggcc”. It cuts these pieces of DNA in different places, because there are differences in the sequences. Only the terminal piece of each different gene is labeled, and they are all different lengths. You then separate these pieces of DNA using electrophoresis. This is done in a gel matrix of agarose or acrylamide. An electric field is set up across the gel and DNA migrates towards the positive pole. Smaller pieces of DNA move faster, and so the fragments are separated by length. The banding pattern is used as a community fingerprint, with each band representing some subset of the organisms in the original sample.
Schematic courtesy of B. Crump

51. Now we know who is there: What next?
Let’s say we have identified an interesting sequence that we want to know more about in the column and quantify it
FISH
Dot Blot
Q-PCR

52. Head et al. 1998 52

53. Fluorescent In-Situ Hybridization (FISH)
FISH: Another use of probes is quite different. It is called fluorescent in situ hybridization, which unfortunately abbreviates to FISH. With this method you actually use probes to label whole organisms. In one approach cells are immobilized on a microscope slide. The Cell walls of the organisms are made permeable and probes are introduced. The slides are then looked at under a microscope and the fluorescent cells are counted. Another approach is to count the fluorescently labeled cells with a flow cytometer.
Schematic courtesy of B. Crump

54. Fluorescent In-Situ Hybridization (FISH)
FISH: Another use of probes is quite different. It is called fluorescent in situ hybridization, which unfortunately abbreviates to FISH. With this method you actually use probes to label whole organisms. In one approach cells are immobilized on a microscope slide. The Cell walls of the organisms are made permeable and probes are introduced. The slides are then looked at under a microscope and the fluorescent cells are counted. Another approach is to count the fluorescently labeled cells with a flow cytometer.
Schleper et al. 2005
Schematic courtesy of B. Crump

55. Schematic courtesy of B. Crump
Dot Blots: One of the most common use of probes is called Dot Blot Hybridization. DNA is extracted from a natural sample and is serially diluted. The DNA is then attached to a membrane in dots. Probes are then introduced to these dilution series dots. The first row of dots was exposed to a general bacteria probe, and the second row of dots was exposed to a probe specific for the proteobacteria. We used factor of ten dilutions so proteobacteria compose approximately 10% of the bacteria in the original sample. Sulfate reducers are approximately 1% of the bacteria in the original sample.
Schematic courtesy of B. Crump

56. 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

57. Quantitative (Real Time) PCR
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

58. Quantitative PCR

59. 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

60. Metagenomics a.k.a., Community Genomics, Environmental Genomics Does not rely on Primers or Probes (apriori knowledge)!
Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS
Image courtesy of John Heidelberg

61. Metagenomics
Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS

62. Metagenomics
Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS

63. Access genomes of uncultured microbes:
Metagenomics
Access genomes of uncultured microbes:
Functional Potential
Metabolic Pathways
Horizontal Gene Transfer …
Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS

64. 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

65. … to the potentially most diverse!
Venter et al. 2004
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!

66. 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

67. 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
Analzye genes from environment

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

69. Access expressed genes of uncultured microbes
Metatranscriptomics
Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS
Access expressed genes of uncultured microbes

70. Schematic courtesy of B. Crump

71. (Some) Problems with Molecular Methods
DNA extraction
Incomplete sampling
Resistance to cell lysis
Storage
Enzymatic degradation
PCR
Inhibitors in template DNA
Amplification bias
Gene copy number
Fidelity of PCR
Differential denaturation efficiency
Chimeric PCR products
Anytime
Contamination w/ non-target DNA

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

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