Download Impacts of DNA-based technologies and PCR basics

Discover the Microbes Within:
Impacts of DNA-based
technologies and PCR basics
Bill Reznikoff
Marine Biological Laboratory
Woods Hole, MA
The Old Way
Classical microbiology – phenotypic approach
Gram stain
Culture
Shapes
The old, old way: Darwin and the first tree…
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
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
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
16S rRNA conservation (red)
Ribosomal RNA (rRNA) Gene Sequencing and Phylogeny
Universal PCR
primers for 16S
rRNA
Figure 2.16
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
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.
Evolutionary Tree of Life Based on 16S and 18S rRNA
ARCHAEA
BACTERIA
Everything we can see
EUKARYA
Carl Woese, 1977
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
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!
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
Correlation Between Genome Size and ORF Content
Figure 13.1
Bacterial Lifestyle Correlates with Genome Size
Variation
Mutational process in bacterial genes is biased
towards deletions
Intracellular lifestyle alters genome size:
DNA Inflow
DNA Outflow
Eukaryotic
cell
Diagram modified from Mira, A., Ochman, H. & Moran, N.A.
2001. Trends Genet 17, 589-96.
Genome Reduction
The process by which a genome decreases in size relative to its
ancestor
• Usually coupled with symbiotic, intracellular lifestyle
• Organelles and endosymbionts
Endosymbionts have the smallest bacterial genomes known
TotalGeneNumber
10000
Wolbachia (1.27 Mb)
9000
8000
7000
6000
5000
*
4000
3000
2000
1000
0
1
16
31
46
61
76
91 106 121 136 151 166 181 196 211 226 241 256 271 286 301 316 331 346 361 376
Bordenstein, unpublished
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
To studying the Wolbachia within?
Credit: Mark Taylor
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
DNA Polymerase Action
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.
PCR - Ready Beads
Small quantity of DNA
Primers
Buffered solution containing DNA
polymerase
DNA polymerase
Four base pairs of DNA
Cofactor MgCl2
All in test tube
Temperature drives the reaction
Target gene:
16S rDNA of Wolbachia
PCR Animation
http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
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
70C hot springs in Yellowstone National Park
DNA thermal cyclers – a
computer that controls repetitive
temperature changes required
for PCR
Example of a thermal cycler from MJ Research
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
Our goal: Determine which of your insects
harbor Wolbachia?
DNA extraction:
PCR:
Gel electrophoresis: