Discover the Microbes Within: Impacts of DNA-based technologies and PCR basics

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Discover the Microbes WithinImpacts of
DNA-based technologies and PCR basics

• Bill Reznikoff
• Marine Biological Laboratory
• Woods Hole, MA

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The Old WayClassical microbiology phenotypic
approach
Gram stain
Culture
Shapes
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The old, old way Darwin and the first tree
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Genes to Cells (Cells to Organisms)

• DNA is the genetic material
• DNA encodes RNA
• RNA (mRNA) encodes proteins
• Proteins (and some RNAs) compose cells
• Cells compose organisms

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The New way The Evolutionary Tree of Life

• Evolution
• The process of genetic change in a line of
  descent over time that results in new varieties
  and species of organisms
• Phylogeny
• Evolutionary relationships between organisms
• Relationships can be deduced by comparing genetic
  information (nucleic acid or amino acid
  sequences) in the different specimens
• Ribosomal RNA (rRNA) are excellent molecules for
  determining phylogeny Carl Woese 1st person to
  use it
• Can visualize relationships on a phylogenetic
  tree

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16S rRNA (ribosomal RNA)

• Small ribosomal subunit involved in mRNA
  translation process
• Ancient molecule, conserved function,
  universally distributed
• Helps identify unknown bacterium to genus or
  species levels
• Present in bacteria eukaryote has very
  divergent copy that is named 18S rRNA present in
  all cells
• Plays a catalytic and structural role in the
  ribosome

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16S rRNA conservation (red)
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Ribosomal RNA (rRNA) Gene Sequencing and Phylogeny
Universal PCR primers for 16S rRNA
Figure 2.16
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Microbial Systematics genetic approach

• Gene sequences are identifiers of organisms
• More accurate, objective, and reproducible
  results
• Identifies unculturable bacteria (99)
• Eliminates special growth requirements
• DNA sequence data are more easily shared and
  databased
• Rapid diagnosis
• Resolves evolutionary relationships through trees
  metaphors for evolutionary diversification

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The Evolutionary Tree of Life

• Comparative rRNA sequencing has defined three
  distinct lineages of cells called domains.
• Bacteria (prokaryotic)
• Archaea (prokaryotic)
• Eukarya (eukaryotic)
• Archaea and Bacteria are NOT closely related.
• Archaea are more closely related to Eukarya than
  Bacteria.

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Evolutionary Tree of Life Based on 16S and 18S
rRNA
ARCHAEA
BACTERIA
EUKARYA
Carl Woese, 1977
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Bacteria Fun Facts

• Most microbes do not cause disease!
• 1 gram of soil 1 billion bacteria
• More bacteria in your mouth than there are people
  in the world
• beneficial bacteria in human intestine of
  cells in human body
• Your colon has the highest density of microbes
  recorded for any microbial habitat
• Oldest forms of life on earth are bacteria (3.8
  billion years ago)
• Bacteria make up most of the biomass on earth,
  but only 1 have been cultured

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Genes and Genomes fun facts

• Bacteria have small genomes (1-7 million base
  pairs vs. 3 billion base pairs in human)
• 1995 First bacterial genome sequence, of
  Haemophilus influenzae
• Two years later 12 genomes sequenced
• April, 2010 4900 prokaryotic genomes sequenced
  or in progress. Wow!

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Prokaryotic Genomes Sizes and Gene Contents

• Rule of thumb A prokaryotic gene 1,000 bp long
• As genome size increases gene content
  proportionally increases
• Prokaryotic genomes range in size from those of
  large viruses to those of eukaryotic microbes
• Unlike eukaryotes, prokaryotic genomes do not
  contain a large fraction of non-coding DNA

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Correlation Between Genome Size and ORF Content
Figure 13.1
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Bacterial Lifestyle Correlates with Genome Size
Variation
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Mutational process in bacterial genes is biased
towards deletions
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Intracellular lifestyle alters genome size
DNA Inflow
DNA Outflow
Diagram modified from Mira, A., Ochman, H.
Moran, N.A. 2001. Trends Genet 17, 589-96.
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Genome Reduction

• The process by which a genome decreases in size
  relative to its ancestor
• Usually coupled with symbiotic, intracellular
  lifestyle
• Organelles and endosymbionts

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Endosymbionts have the smallest bacterial genomes
known

Bordenstein, unpublished
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How do we go from here..?
Crustaceans (35)
Insects (20-75!)
Filarial nematodes (90)
Chelicerates
2-6 million insect species are infected with
Wolbachia!!
Arthropods
Nematodes
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To studying the Wolbachia within?
Credit Mark Taylor
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PCR (Polymerase Chain Reaction)

• 1- Amplifies defined DNA sequences
• 2- Uses basic properties of the DNA replicating
  enzyme (DNA Polymerase)
• 3- DNA polymerization using defined DNA template,
  two defined primers, 4 dNTPs

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DNA Polymerase Action
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Basics of PCR

• Template DNA - the starting DNA of interest.

• High temperature denatures template DNA into
  single strands and synthetic sequences of ssDNA
  (20-30 nucleotides) serve as primers

• Two different primers are used to bracket the
  target gene to be amplified

• DNA polymerase copies the complimentary strand
  starting at the primer. In one cycle, two
  identical strands are made.

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PCR - Ready Beads

• Small quantity of DNA
• Primers
• Buffered solution containing DNA polymerase
• Four base pairs of DNA
• Cofactor MgCl2

All in test tube

• Small quantity of DNA
• Primers

• Buffered solution containing DNA polymerase
• Four base pairs of DNA
• Cofactor MgCl2

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Temperature drives the reaction
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Target gene 16S rDNA of Wolbachia
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PCR Animation
http//highered.mcgraw-hill.com/olc/dl/120078/micr
o15.swf
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Two Key Innovations for Success of PCR

• Heat-stable DNA polymerase isolated from
  bacterium Thermus aquaticus which inhabits hot
  springs
• Polymerase remains active despite being heated
  many times
• DNA thermal cyclers a computer that controls
  repetitive temperature changes required for PCR

70C hot springs in Yellowstone National Park
Example of a thermal cycler from MJ Research
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PCR and Pop Culture

• Jurassic Park and CSI
• Some fun PCR facts to share with your students
  PCR has been used to amplify DNA from
• a preserved quagga (a zebra relative that became
  extinct 100 years ago)
• crime scenes (e.g., O.J.)
• Abducted children to find parents
• Prisoners leading to exonerations
• Mummies to determine gender, bacterial infections

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Our goal Determine which of your insects harbor
Wolbachia?

• DNA extraction

PCR
Gel electrophoresis
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