Transcript Impacts of DNA-based technologies and PCR basics

Discover the Microbes Within:
Impacts of DNA-based
technologies and PCR basics
Seth Bordenstein
Marine Biological Laboratory
April 11, 2008
Microbes in Eukaryotic Evolution
ARCHAEA
BACTERIA
EUKARYOTE
Everything we can see
C. Woese
Bacteria Fun Facts
Most microbes do not cause disease!
1 gram of soil = 10 million bacteria
More bacteria in your mouth than there are
people in the world
# beneficial bacteria in human intestine = # of
cells in human body
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
Classical microbiology – phenotypic approach
Gram stain
Culture
Shapes
Molecular microbiology – genotypic approach
Identifies genes
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
Genes and Genomes: fun facts
Bacteria have small genomes (110 million base pairs vs. 3 billion
base pairs in human)
1995: Entire genome of
Haemophilus influenzae was
sequenced
Two years later: 12 genomes
sequenced
March 24, 2008: 747 genomes
sequenced (613 are from bacteria)
and 1753 ongoing bacterial
genomes
Buchnera (0.64 Mb)
Blochmannia (0.75 Mb)
Wigglesworthia (0.70 Mb)
Wolbachia (1.27 Mb)
Endosymbionts are the smallest bacterial genomes known
Insect endosymbionts
Wernegreen 2002
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 Introduction
Polymerase Chain Reaction (PCR) allows scientists to amplify
minutes amount of a specific DNA sequence from a
heterogenous DNA pool in a few hours
Invented by Dr. Kary Millis in 1983 (Nobel Prize in Chemistry in
1993)
One of the most widely used biotechnology techniques in
biological research. PCR is the method of choice for symbiont
detection.
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.
To perform your 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
Small ribosomal subunit involved in mRNA translation process
Ancient molecule, functionally constant, universally distributed
Helps identify unknown bacterium to genus or species levels
Present in bacterial symbionts; eukaryote has very divergent
copy that is named 18S rRNA
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.)
eight-celled human preembryos, to
detect cystic fibrosis
the brain of a 7000 year old
American mummy
patients for disease diagnosis
Our goal: Determine which of your insects
harbor Wolbachia?
DNA extraction:
PCR:
Gel electrophoresis:
Bellingham HS, MA Falmouth HS, MA
Bronx HS, NY
One day till we discover the
microbes within!
The Wolbachia Project: A “Bridge”
Science in the Classroom:
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
2007-2008
Student
2005
Workshop
Participant
to New
Cro ss Type
Infe cted Male
x
Uninfected Female
Infe cted Male
x
Infe cted Female
Uninfected Male
x
Uninfected Female
Uninfected Male
x
Infe cted Female
ID #
Fly
Gender
1
F
+
-
-
2
F
+
-
-
3
F
+
-
-
6
M
+
-
-
7
M
+
-
-
8
M
+
-
-
11
F
+
+
+
12
F
+
+
+
13
F
+
+
+
16
M
+
+
+
17
M
+
+
+
18
M
+
+
+
21
F
+
-
-
22
F
+
-
-
23
F
+
-
-
26
M
+
-
-
27
M
+
-
-
28
M
+
-
-
31
F
+
+
+
32
F
+
+
+
33
F
+
+
+
36
M
+
+
+
37
M
+
+
+
38
M
+
+
+
Insect
DNA
PCR Results
Wolbachia Phage
DNA
DNA
Notation Key
(+) - positive result
(-) - nega tive result
*Results suggest all samples have insect DNA. Results suggest samples with an infected
mother have Wolbachia and the virus. Due to an error in the dilution of the Wolbachia
primer, research was conduc ted twice.
Senior Student
North Attleboro High
School, MA
“A Safer Alternative to
Traditional Insecticides:
Exploring the Applications
of Wolbachia”
 submitted to the JSHS
Symposium at Boston
University
 selected to create a
poster of her research
 won an award for being
the most creative project at
the fair
 won first place at the
regional science fair
 continues on to the state
science fair in May
Progression of the Wolbachia Project
New Science
Professional
Development
Implementation
Summer
Envisionships