3. 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
5. 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.
6. PCR - Ready Beads
• Small quantity of DNA
• Primers
• Buffered solution containing
DNA polymerase
• Four base pairs of DNA
• Cofactor MgCl2
All in test tube
Buffered solution containing DNA
polymerase
Four base
Cofactor MgCl2
Small quantity of DNA
Primers
7. 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
10. Importance of Ribosomes in Cell Biology
Ribosomes are part of the machinery
that synthesizes cell proteins
Ribosomes are so important that they
are ubiquitous and highly conserved
11. Secondary structure
of 16S rRNA in E. coli
Molecular
Phylogenetics
Step 1. Select a DNA
region that is homologous,
or similar across species
due to common ancestry.
Ribosomal RNA (rRNA)
Ideal gene for phylogenetic
studies because it :
• is an essential gene that is
present in all organisms.
• is a common target for
sequencing studies; large
database for comparisons.
• contains sites that are
relatively conserved (stems)
and sites that are more free to
vary (loops).
12. Our goal: Determine which of your insects
harbor Wolbachia?
DNA extraction: PCR: Gel electrophoresis:
13. 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
14. How is this organism related to other species?
A
F
D
E
B
H
I
J
K
?
G
C
15. DNA sequences provide characters that are:
• Numerous, discrete characters (A, T, C, G)
• Directly comparable across species
• Unlikely to change due to culture conditions
• Can be sampled without ever culturing (important for endosymbionts!)
ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAG
GAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGACAGATGTCTTGTAATCC
GGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGT
GAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAA
CACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTA
GCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGG
GAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAG
CTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTCAATACGCGTGCTCAGGCAGATAAAATATTA
Molecular phylogenetics:
Inference of evolutionary relationships based on molecular data
16. 2. Amplify and Sequence this region across isolates….
ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT
AGGGAAAGGACATTTAGTGAAAGAAATTGATG
CGATGGGTGGATCGATGGCTTATGCTATCGATC
AATCAGGAATTCAATTTAGAGTACTTAATAGTA
GCAAAGGAGCTGCTGTTAGAGCAACACGTGCT
CAGGCAGATAAAATATTATATCGTCAAGCAATA
CGT
ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT
AGGGAAAGGACATTTAGTGAAAGAAATTGATG
CGATGGGTGGATCGATGGCTTATGCTATCGATC
AATCAGGAATTCAATTTAGAGTACTTAATAGTA
GCAAAGGAGCTGCTGTTAGAGCAACACGTGCT
CAGGCAGATAAAATATTATATCGTCAAGCAATA
CGT
ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT
AGGGAAAGGACATTTAGTGAAAGAAATTGATG
GTFTGGGTGGATCGATGGCTTATGCTATCGATC
AATCAGGAATTCAATTTAGAGTACTTAATAGTA
GCAAAGGAGCTGCTGTTAGAGCAACACGTGCT
CAGGCAGATAAAATATTATATCGTCAAGCAATA
CGT
ACAGATGTCTTGTAATCCGGCCGTTGGTGGCAT
AGGGAAAGGACATTTAGTGAAAGAAATTGATG
CGATGGGTGGATCGATGGCTTATGCTATCGATC
AATTTAGAATTCAATTTAGAGTACTTAATAGTAG
CAAAGGAGCTGCTGTTAGAGCAACACGTGCTC
AGGCAGATAAAATATTATATCGTCAAGCAATAC
GT
Sequence the
PCR product
PCR
17. 3. Estimate relationships based on extent of DNA similarity.
G
B
C
D
A
J
F
E
K
H
I
ATGTTGGCAGTCCGATGTAAGC
ATGTTGGCAGTCCGATGTAAGC
ATGTTGGCAGTCCGATGTAACC
ACGGTAGCAGTCTGATGTATCC
ACGGTAGCAGTCTGATGTATCC
ACGGTAGCAGTCTGATGTATCC
CTGCTGGTAGTCGTTTGTAACC
CTGCTGGTAGTCGTTTGTAACC
CTGCTGGCAGTCGGTTGTAACC
ATGCTGGCAGTCGGGTGTAACC
ATGGTGGCAGTCGGGTGTCACC
Colored letters = different from top sequence (taxon G)
Molecular phylogeny of taxa A-I.
At variable DNA positions, related
groups will tend to share the
same nucleotide.
The sheer number of characters is
helpful to distinguish the
‘phylogenetic signal’ from noise.
18. The elegant idea behind DNA sequencing
Technology changes quickly, but for many years we’ve used Sanger’s cool trick.
Fred Sanger
In the 1970’s, Sanger’s group discovered a fundamentally new method of
'reading' the linear DNA sequence using special bases called chain terminators or
dideoxynucleotides.
This method is still in use today.
What is the basis of Sanger’s method?
Shared with Walter Gilbert and Paul Berg
25. Fractionating DNA Molecules
DNA is negatively charged at
neutral pH
DNA will travel towards a
positive charge
DNA electrophoresed in a gel
will travel according to its chain
length
26. Detecting DNA
Early experiments used radio-
isotopically labeled DNA
(primers)
Now we use fluorescently
labeled dideoxynucleotides
28. Use of capillaries filled with a matrix for higher
through-put of sequencing reactions.
e.g. ABI 3730
29. 1. Purify PCR
product
2. Set up sequencing
reaction
3. Perform
cycle
sequencing
4. Resolve sequence
fragments
5. Read order of
terminators (DNA
sequence)
In sum: From PCR to sequence data
30. Quality of sequence data may vary, depending on:
• Purity and concentration of template DNA
• Presence of extra PCR bands (artifacts)
• Quality of dye-terminators, electrophoresis matrix, and other reagents
Ideally, look at chromatograms and convince yourself that base
calls are robust.
31. DNA sequence assembly: Combining sequence reads to
build the entire sequence of the template DNA
PCR PRODUCT of 1,200 bp
Sequence read #1
Sequence read #2
Sequence read #3
Sequence “reads” typically range from 500-800 bp. Often the total DNA fragment is much longer.
Regions of overlap among sequence reads:
• Let us piece together the linear “puzzle” of the template DNA sequence.
• Often confirm base calls and improve overall add data quality.
32. Major sequencing centers have facilities to sequence
multiple full genomes each year
Due to genomic research over the past decade, DNA sequencing has
become:
• Increasingly automated
• Higher-throughput
• Less $$ per reaction
• And (good news) more accessible to researchers and educators
33. Many public universities have DNA sequencing facilities in house.
Such facilities are usually able to run small numbers of reactions for a
cost of ~$10-15 per reaction, but often much less for “internal users”
with ties to the university.
Ideal situation: Team up with faculty at your local university to act as
facilitators.
Possible avenues for DNA sequencing:
Even if DNA sequencing not an option for classroom labs, important to
discuss:
• how DNA sequencing is performed,
• how data is evaluated,
• what information can be obtained from the data,
• what biologically important concepts can be learned,
• ethical issues surrounding its interpretation and use.
34. Ethical issues involving DNA sequencing
Truro: A tranquil Cape community shaken by the murder of a young woman in
January 2002
36. Thylacine pup preserved in alcohol in
1866. Its cells could be used for
cloning. By chance this Thylacine was
stored in a jar of alcohol rather than
formalin, which would have destroyed
the DNA.
Should scientists attempt to clone the now-extinct Thylacine to revive
this species?? (The technology will be there eventually.)
