Molecular Systematics

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James Joyce
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Paul Allen
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Leroy Hood
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Molecular Systematics
Judd et al pp. 103-118
The use of DNA and RNA sequences
to infer evolutionary relationships
Why Introduce Molecular Systematics?
• So you gain a basic understanding of the tools
available, what they can and can’t offer, and
how they work
• To provide you with the vocabulary and
concepts used by molecular systematists
• NOT to teach you how to go into a lab and
start doing the work
• It’s the wave of the present and future
Arabidopsis thaliana: first plant
genome to be sequenced.
Sequencing began
in 1996 and was
completed in 2000.
125 Mbp (=125
million base pairs!)
DNA sequencing techniques are driven by speed and cost
Major landmarks in DNA sequencing
1953 Discovery of the structure of the DNA double helix.[49]
1972 Development of recombinant DNA technology, which permits isolation of defined fragments of DNA; prior to this, the only accessible
samples for sequencing were from bacteriophage or virus DNA.
1977 The first complete DNA genome to be sequenced is that of bacteriophage φX174.[50]
1977 Allan Maxam and Walter Gilbert publish "DNA sequencing by chemical degradation".[5] Frederick Sanger, independently, publishes "DNA
sequencing with chain-terminating inhibitors".[51]
1984 Medical Research Council scientists decipher the complete DNA sequence of the Epstein-Barr virus, 170 kb.
1986 Leroy E. Hood's laboratory at the California Institute of Technology and Smith announce the first semi-automated DNA sequencing
1987 Applied Biosystems markets first automated sequencing machine, the model ABI 370.
1990 The U.S. National Institutes of Health (NIH) begins large-scale sequencing trials on Mycoplasma capricolum, Escherichia coli, Caenorhabditis
elegans, and Saccharomyces cerevisiae (at US$0.75/base).
1991 Sequencing of human expressed sequence tags begins in Craig Venter's lab, an attempt to capture the coding fraction of the human
1995 Craig Venter, Hamilton Smith, and colleagues at The Institute for Genomic Research (TIGR) publish the first complete genome of a freeliving organism, the bacterium Haemophilus influenzae. The circular chromosome contains 1,830,137 bases and its publication in the journal
Science[53] marks the first use of whole-genome shotgun sequencing, eliminating the need for initial mapping efforts.
1996 Pål Nyrén and his student Mostafa Ronaghi at the Royal Institute of Technology in Stockholm publish their method of pyrosequencing[54]
1998 Phil Green and Brent Ewing of the University of Washington publish “phred” for sequencer data analysis.[55]
2000 Lynx Therapeutics publishes and markets "MPSS" - a parallelized, adapter/ligation-mediated, bead-based sequencing technology, launching
"next-generation" sequencing.[56]
2001 A draft sequence of the human genome is published.[57][58]
2004 454 Life Sciences markets a parallelized version of pyrosequencing.[59][60] The first version of their machine reduced sequencing costs 6-fold
compared to automated Sanger sequencing, and was the second of a new generation of sequencing technologies, after MPSS. [29]
Molecular Data
• Many more molecular characters available for
analysis than morphological ones.
• Identity is easier to define: ATCG vs. whether a
flower color is pink or white.
• Nonetheless, molecular data are still subject to
homoplasy: reversals and convergence as well as
long branch attraction (errors due to mutation
rate being fast and number of characters small:
leads to wrong phylogenetic tree appearing to be
For example, two plants may have a
“C” at a particular location on a gene
• One possibility is that they have evolved
together and are closely related
• Another possibility is that one started at with
the “C” at that location and it didn’t change,
while the other plant went “C->G->A->T->C”
and looks like it’s the same evolution because
all you see is the start and finish “C”
Modern Phylogenetics
• In spite of the pitfalls, “DNA sequence data
are now overwhelmingly the tool of choice for
generating phylogenetic hypotheses.” from
J&C, pg. 103
• Much of this data is on the web.
• National Center for Biotechnology Information
Nucleotide Structure– Phosphate
group, sugar and nitrogenous base
Hooks up with
the position 3’
OH group on
the next
**Required to
hook nucleotides
together in the
making of DNA
Hence “deoxy-” in
Structure of DNA
Structure of DNA
Plant Genomes
• Plants contain three different genomes:
chloroplast, mitochondrial, nuclear.
• The chloroplast & mitochondrial genomes
were acquired from algae or bacteria millions
of years ago.
• All three genomes are used in molecular
Nuclear, Chloroplast, Mitochondrial
Genomes in Comparison
Genome Size
More stable than
mitochondrial genome
Rearrangements occur
so often as to make
not useful frequently
135-160 (small) Cyanobacteria
(sometimes via
an alga)
(Seed parent)
maternal (Seed
Over a million
Genetic history
not same as
species history
Systematists use data from all three of these
Chloroplast Genome (circular)
• Stable within cells and species (more so than
mitochondrial genome)
• Large Single Copy (LSC), Small Single Copy
(SSC) and Inverted Repeat (IRa & IRb regions)
• Introns– noncoding regions between coding
regions (exons) Gains and losses of genes and
their introns are phylogenetically useful.
• Rearrangements of the chloroplast genome
demarcate major groups.
Chloroplast Genome: Vitis vinifera
LSC= large
single copy
SSC= Small
single copy
Q: Why does this look like a circular genome?
Each Gene Mutates at a Different Rate
• Genes coding for vital enzymes or structures
tend to be more conserved.
• The frequency of a mutation of a gene
determines its utility for addressing a specific
• Slow rate of mutation– used for older groups
• Fast rate of mutation– used to assess
relationships in closely related populations
Gene Mutation Rate Problems
• If a gene is mutating very slowly, the level of
variation approaches the sequencing error rate
and inferences become unreliable
• If a gene is mutating very quickly, parallelisms and
reversals accumulate so fast that all phylogenetic
information is lost
• Genes have to be picked for a given study based
on what information is desired and what rate of
genetic mutation will be required for that goal.
Methods in Molecular Systematics
• Allozyme fingerprinting: different alleles
produce slightly different proteins which migrate
differently on an electrically charged gel. Takes
about 4 hours per gel, but up to about 30
samples can be run at once. An older method,
but less than $100/run.
• DNA sequencing– expensive but cost coming
down considerably. Much of the process has now
been automated. The wave of the future is here!
Allozyme Fingerprinting– older
method but can still be useful
• Uses common enzymes to look for differences, e.g.,
Malate Dehydrogenase (MDH) and
Phosphoglucomutase (PGM) (G1P to G6P and back
• Less automated, older method but still useful when
exact sequence is not necessary– e.g., differentiating
two closely related species of one genus
• (Variant forms of an enzyme that are coded by
different alleles at the same locus are called allozymes.
These are opposed to isozymes, which are enzymes
that perform the same function, but which are coded
by genes located at different loci.)
Allozyme Fingerprinting
DNA Sequencing– has always been limited by
small amount of DNA available for sequencing
• Older method: Polymerase Chain Reaction
(PCR) to make huge amounts of DNA followed
by Restriction Site Analysis. Best for
ordering sequence of genes on a
• Newer method: use dideoxynucleotides and
read colors as they come off the machine!
Complete genome sequencing.
Polymerase Chain Reaction
the primer
is the hard
part– you
have to
about the
gene you
want to
ahead of
Restriction Site Analysis
(after you do PCR to get enough material)
• Restriction Enzymes cut DNA at particular
sequence of nucleotides.
• Use one restriction enzyme, then another,
then both together and you can puzzle out the
order of the restriction sites by fragment size.
• Useful to find order of genes on chromosome
• Can cover large stretches of DNA at a time
Automated Gene Sequencing
• See:
• “We can get the sequence of a fragment of DNA as long as
900 or so nucleotides. Great! But what about longer
pieces? The human genome is 3 *billion* bases long,
arranged on 23 pairs of chromosomes. Our sequencing
machine reads just a drop in the bucket compared to what
we really need! To do it, we break the entire genome up
into manageable pieces and sequence them.”
• Cooperative efforts are necessary to sequence large
Automated Gene Sequencing
DNA sequencing reactions are just like the PCR
reactions for replicating DNA (refer to the
previous page DNA Denaturation, Annealing
and Replication). The reaction mix includes the
template DNA, free nucleotides, an enzyme
(usually a variant of Taq polymerase) and a
'primer' - a small piece of single-stranded DNA
about 20-30 nt long that can hybridize to one
strand of the template DNA. The reaction is
initiated by heating until the two strands of
DNA separate, then the primer sticks to its
intended location and DNA polymerase starts
elongating the primer. If allowed to go to
completion, a new strand of DNA would be the
result. If we start with a billion identical pieces
of template DNA, we'll get a billion new copies
of one of its strands.
Automated Gene Sequencing
Dideoxynucleotides: We
run the reactions, however,
in the presence of a
This is just like regular
DNA, except it has no 3'
hydroxyl group - once it's
added to the end of a DNA
strand, there's no way to
continue elongating it.
Now the key to this is that
MOST of the nucleotides
are regular ones, and just a
fraction of them are
dideoxy nucleotides....
Automated Gene Sequencing
Replicating a DNA strand in the presence of
dideoxy-T MOST of the time when a 'T' is
required to make the new strand, the enzyme
will get a good one and there's no problem.
MOST of the time after adding a T, the enzyme
will go ahead and add more nucleotides.
However, 5% of the time, the enzyme will get a
dideoxy-T, and that strand can never again be
elongated. It eventually breaks away from the
enzyme, a dead end product.
Sooner or later ALL of the copies will get
terminated by a T, but each time the enzyme
makes a new strand, the place it gets stopped
will be random. In millions of starts, there will
be strands stopping at every possible T along
the way.
ALL of the strands we make started at one
exact position. ALL of them end with a T. There
are billions of them ... many millions at each
possible T position. To find out where all the
T's are in our newly synthesized strand, all we
have to do is find out the sizes of all the
terminated products!
Automated Gene Sequencing
Here's how we find out those fragment sizes. Gel electrophoresis can be
used to separate the fragments by size and measure them. In the cartoon at
left, we depict the results of a sequencing reaction run in the presence of
dideoxy-Cytidine (ddC).
First, let's add one fact: the dideoxy nucleotides in my lab have been
chemically modified to fluoresce under UV light. The dideoxy-C, for example,
glows blue. Now put the reaction products onto an 'electrophoresis gel' (you
may need to refer to 'Gel Electrophoresis' in the Molecular Biology Glossary),
and you'll see something like depicted at left. Smallest fragments are at the
bottom, largest at the top. The positions and spacing shows the relative sizes.
At the bottom is the smallest fragment that's been terminated by ddC; that's
probably the C closest to the end of the primer (which is omitted from the
sequence shown). Simply by scanning up the gel, we can see that we skip two,
and then there's two more C's in a row. Skip another, and there's yet another
C. And so on, all the way up. We can see where all the C's are.
Automated Gene Sequencing
Putting all four deoxynucleotides into the
picture: Well, OK, it's not so easy reading
just C's, as you perhaps saw in the last
figure. The spacing between the bands
isn't all that easy to figure out. Imagine,
though, that we ran the reaction with *all
four* of the dideoxy nucleotides (A, G, C
and T) present, and with *different*
fluorescent colors on each. NOW look at
the gel we'd get (at left). The sequence of
the DNA is rather obvious if you know the
color codes ... just read the colors from
bottom to top: TGCGTCCA-(etc).
(Forgive me for using black - it shows up
better than yellow)
Automated Gene Sequencing
An Automated sequencing gel: That's exactly what we do
to sequence DNA, then - we run DNA replication reactions
in a test tube, but in the presence of trace amounts of all
four of the dideoxy terminator nucleotides. Electrophoresis
is used to separate the resulting fragments by size and we
can 'read' the sequence from it, as the colors march past in
In a large-scale sequencing lab, we use a machine to run
the electrophoresis step and to monitor the different
colors as they come out. Since about 2001, these machines
- not surprisingly called automated DNA sequencers - have
used 'capillary electrophoresis', where the fragments are
piped through a tiny glass-fiber capillary during the
electrophoresis step, and they come out the far end in
size-order. There's an ultraviolet laser built into the
machine that shoots through the liquid emerging from the
end of the capillaries, checking for pulses of fluorescent
colors to emerge. There might be as many as 96 samples
moving through as many capillaries ('lanes') in the most
common type of sequencer.
At left is a screen shot of a real fragment of sequencing gel
(this one from an older model of sequencer, but the
concepts are identical). The four colors red, green, blue
and yellow each represent one of the four nucleotides.
The actual gel image, if you could get a monitor large
enough to see it all at this magnification, would be perhaps
3 or 4 meters long and 30 or 40 cm wide.
Most Studied Gene Sequences
• Rubisco (from chloroplast, rbcL)
• Ribosome subunits (from nucleus, 18S & 26S)
• ATP synthase (from chloroplast, atpB)

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