|
The discoveries of the genetic revolution
have provided mycologists with extremely powerful tools to analyse
the biology of their chosen fungus, and to research the role of
fungi in nature, disease and biotechnology. Let’s look at
some of the key methods used to explore fungal molecular biology:
| i) |
The Polymerase Chain
Reaction: Amplifying DNA for forensics, fungal research or
for medicine
The Polymerase Chain Reaction (PCR) is a
process that uses a heat-resistant DNA polymerase
to generate billions of copies from even single copies of
DNA! Let’s see how scientists do it: |
| |
|
|
| |
© Arwyn Edwards (this photo
and all others on this page) |
|
First, the DNA needs to be extracted
from a sample of cells. This is done by breaking the cells
open with a detergent and precipitating the DNA. |
|
| |
|
|
| |
Next, we need to prepare
a mix of the chemicals and DNA polymerase needed to do the PCR.
The recipe includes a buffer, magnesium chloride (helps the
polymerase to work), the four nucleotides, two primers
which mark the sequence of DNA to be copied, and a heat-stable
DNA polymerase called HTaq (from bacteria that
live in hot springs). The whole mix is diluted with water. Throughout,
great care is taken to avoid contamination with stray DNA from
the lab or the scientist doing PCR. |
| |
|
| |
|
|
|
| |
Once the reactants have been
mixed, and some of the DNA sample added, the reaction is placed
in a thermal cycler, which is programmed to separate the DNA
strands by heating, then cool to allow the primers to bind,
and then warm to the optimal temperature for the HTaq to fill
in the gaps between the primers:
Each cycle of melting, binding and synthesizing
doubles the number of specific DNA fragments. If the process
is allowed to continue for 20, 30 or 40 cycles, the number
of fragments generated is astronomical. In effect, PCR turns
a needle in a haystack into a haystack of needles!
|
| |
Once the PCR is complete,
we can take some of the reaction mixture and analyse it
using gel electrophoresis:
|
|
 |
|
| |
|
| |
| This process separates DNA
fragments by length. The phosphate groups on the DNA molecule
are negatively charged, so they are drawn towards a positive
electrode (cathode) when in an electric field. If we put
in a gel (usually made from agarose or polyacrylamide)
this slows down bigger DNA fragments more than smaller
fragments: |
|
 |
|
| |
|
| |
Once we turn off the electric field,
we can stain the gel with a glowing chemical, which shows us
where the DNA bands are in the gel when we shine ultraviolet
light on the gel: |
| |
|
| |
|
| |
|
| |
We can use “ladders”
of DNA fragments of increasing size to tell us how large our
DNA fragments are. The “ladder” displayed above
increases in 100 base-pair increments. If our PCR has worked
we can spot fragments of specific sizes on the gel. Here,
we can see the second sample contained our target DNA, and
that it is about 600 base pairs in size:
Once we've shown that our PCR
worked, we can use the DNA we have made for cloning,
DNA fingerprinting or DNA sequencing.
In medical mycology, the presence
of a band in the right place is often evidence of a specific
fungal pathogen. Carefully designed PCRs are often very specific
and highly sensitive to the extent that the scientist can
just print a picture of the gel and leave it at that. |
| |
|
| ii) |
Restriction Mapping
and DNA Fingerprinting
Certain enzymes, called restriction
enzymes, cut DNA at specific restriction
sites. For the enzyme HindIII (the third
restriction enzyme isolated from Hemophilus influenzae
strain d) the restriction site is:
5’-AAGCTT-3’
3’-TTCGAA-5’
If we have a DNA fragment of a certain size,
perhaps isolated by PCR, we can use restriction
enzymes to create a map of that fragment, by looking at where
these sites occur using the enzymes and gel electrophoresis
to separate the cut DNA. This is called restriction
mapping. |
| |
|
| |
| The first step is to add some enzyme
and buffer to the DNA samples and dilute the mix with
water to the right concentration. The enzyme will then
do its job if we warm the reaction to body temperature
for a few hours. |
 |
|
| |
|
| |
Restriction mapping can be used
in DNA fingerprinting by means of a procedure
called Restriction Fragment Length Polymorphism (RFLP).
This is based on the idea that mutations may introduce or
delete restriction sites, or change the relative position
of a site. As mutations are heritable changes in genetic material,
some RFLPs can be indicators of parentage, and as such, some
members of a population may have a restriction site at point
X, while others have it at point Y, and some at point Z. Some
may not have it at all. The different forms are called alleles
and the existence of more than one allele in a population
is called polymorphism. When we look at several
alleles across a complex genome the chance
that two certain individuals will have the same RFLP allele
pattern is minimal. This is aided by the fact that humans
are diploid (having two sets of chromosomes)
and one chromosome may have a different RFLP to the corresponding
chromosome in the other set. All this allelic
variation forms the basis of DNA fingerprinting
which can be used either in taxonomic studies, or more recognizably
in forensic genetics.
However, in a complex genome there will be a great many
restriction sites that aren’t polymorphic and will clutter
up our electrophoresis gel. To filter these out, we can either
use PCR to amplify our target regions to give a better signal,
or use Southern Blotting.
Southern Blotting, named after its inventor,
Professor Ed Southern involves sucking the DNA out of the
gel, baking it on to a nylon membrane, allowing a probe
to bind with any complementary DNA sequences on the membrane-bound
DNA, and then removing spuriously bound probe. The probe can
be labelled with radioactive phosphates, or with fluorescently
tagged DNA. We can then see the position of the probe using
either autoradiographs or fluorescence scanners:
A fluorescent RFLP. The first sample (2nd
lane from the left) has
a restriction site, where the next two lack it. Case closed.
However, RFLP, even when
coupled with PCR, isn’t particularly
good for forensics. Recently, forensic scientists have turned
to an automated method (microsatellite analysis)
that can use microscopic traces of DNA to secure a conviction.
To do this, several fluorescent PCR primer pairs are used
to look at several locations across the genome (loci).
Currently, 14 loci are examined in a special type of PCR called
Single Generation Multiplex PCR (SGM PCR).
| Microsatellite loci aren’t some kind
of new digital TV deal, but small segments of DNA that
are repeated. The most useful microsatellites, Short
Tandem Repeat (STR) loci, are spread throughout
the human genome and look like this: |
| |
5’- CACACACACACACACA -3’ (08
repeats) |
In another person,
this may be: |
| |
5’-CACACACACACACACACACACACA-3’ (12 repeats) |
The number of CA repeats changes the size
of the DNA fragment amplified by SGM PCR
which can then detected by high resolution electrophoresis
and given a number corresponding to the number of repeats.
A 14 locus STR profile may look something like:
(08) (23) (12) (28) (09) (07) (11) (20) (14)
(28) (14) (34) (06) (39).
Given that there are as many as 50 alleles
at each locus, the chance that an individual's
profile exactly matches someone else's (apart from his or
her identical twin), assuming equal frequency of each allele,
is 0.02 to the power 14, that is about 0.0000000000000000000000016384,
give or take a few zeros. This means there is a good degree
of certainty, in most cases, when convictions are made on
the basis of DNA evidence.
One study looked at ten such marker
loci in the human genome, and compared the frequency
of people with the same number of repeats at each locus. Here
are the results for the proportion of Germans and Austrians
tested for one marker, D2S1338:
| D2 D2S1338
- Germans |
D2 D2S1338
- Austrians |
| 15
16
17
18
19
20
21
22
23
24
25
26 |
0.005
0.005
0.180
0.060
0.095
0.125
0.035
0.055
0.105
0.155
0.110
0.025
|
15
16
17
18
19
20
21
22
23
24
25
26 |
None
0.041
0.215
0.090
0.122
0.141
0.060
0.025
0.095
0.095
0.103
0.019
|
|
| |
Population
data on the AmpF/STR SGM plus PCR amplification kit in Germans
and Austrians.
Forensic Science International 132
(2003) 84-86 |
| |
|
| |
As you can see, there are a number
of differences that suggest we could predict the nationality
of someone on the basis of their STR profiles. A recent study
showed the possibility of doing this with Welsh and English
males. |
| |
|
| iii) |
Cloning DNA
Before the days of PCR, if fungal molecular
biologists wanted to get a large amount of DNA, we would use
molecular cloning. This process is still
of use for DNA sequencing and the production
of foreign proteins (see the example on insulin).
To do this, we would treat our target DNA with a restriction
enzyme that would give sticky ends.
If we were to cut a vector with the same enzyme, we would
have complementary sticky ends. We could then seal the target
DNA and the vector together using an enzyme called DNA
ligase.
The vector used varies according to the job, but usually
plasmids (loops of bacterial or yeast DNA
that can replicate themselves outside of the organism’s
main genome) are used. Modern plasmids are artificial constructs,
and contain four key features:
|
| |
| a) |
A replication origin that
works in the host – this is where
DNA replication starts. |
| b) |
A multiple cloning site or polylinker
– this site contains several restriction sites to
allow many restriction enzymes to be used. |
| c) |
Antibiotic resistance gene. This allows
only bacteria containing the plasmid to grow on agar containing
antibiotics (usually antibiotics that are of little use
in medicine). |
| d) |
A colour-producing enzyme gene straddling
the polylinker. Usually, beta galactosidase is used. This
takes a derivative of galactose and modifies it to give
a nice blue colour. Cells with working galactosidase make
blue colonies. However, if the polylinker is broken by
the insertion of the target DNA, the enzyme stops working,
and the colony is white. Therefore, we can tell which
cells contain the plasmid, and which ones contain our
target DNA as well. |
|
| |
|
| |
Once the plasmid is sealed, we
can electrically shock cells (usually either the bacterial species
Escherichia coli or the yeast Saccharomyces cerevisiae)
to introduce the DNA, and following growth on selective nutrient
media (antibiotics and galactose derivative added) we can see
which colonies have both the plasmid and our target DNA. We
can then isolate the plasmid DNA for analysis: |
| |
|
 |
An
agar plate containing genetically modified (GM) E.
coli. The blue colonies are boring, as they only
contain the plasmid, but the white ones have both the
plasmid and the fungal DNA introduced successfully.
|
|
| iv) |
DNA sequencing
Once we have PCR amplified DNA or cloned
DNA we can work out the nucleotide sequence. Nowadays,
fungal molecular biologists tend to use automated, very expensive
machines (£120,000 each, plus postage and packaging)
called sequencers to analyse the products
of sequencing reactions.
The procedure used is modified from that devised by Fred
Sanger at Cambridge in the sixties (Fluorescent Primer Chain
Termination). A fluorescently labelled primer acts to direct
DNA synthesis with a DNA polymerase. This
incorporates the four nucleotides in the same way as PCR -
but, wait! – There’s a catch – or rather
the lack of one!
Normal DNA nucleotides have an alcohol group in the 3’
position. This catches on to the 5’
phosphate of the next nucleotide, allowing the chain of nucleotides
to grow. But Sanger sequence mixes include a small quantity
of nucleotides without the 3’ alcohol (so called dideoxynucleotide
triphosphates – ddNTPs as opposed to deoxynucleotide
triphosphates – dNTPs). This jams the process of DNA
synthesis at the position where the ddNTP is incorporated.
As such, four tubes, primers with four different
fluorescences, and four ddNTP mixes are needed. If we take
the mix that is used to sequence adenine positions, we will
have a primer with a green fluorescent tag, DNA polymerase,
dCTP, dGTP, dTTP, some dATP, and some ddATP. The thymine terminating
mix has a red fluorescent primer, DNA polymerase, dCTP, dGTP,
dATP, some dTTP, and some ddTTP. The pattern is the same for
cytosine and guanine terminating mixes.
All the components of four sequence termination mixes,
apart from fluorescent primers and the template DNA.
The end result is that we have fragments of
DNA of increasing size that always end with the ddNTP nucleotide.
When we mix all four reactions we should have a set of fragments
increasing in one nucleotide increments up to 2000 nucleotides
long. The sequence can then be read using a capillary
electrophoresis sequencer that separates small DNA
fragments using a very high voltage in a buffer-containing
glass tube with extremely high resolution and picks up the
fluorescent primer with a laser scanner. 96 samples can be
analysed within a matter of minutes, and the results displayed
on a PC:
 Sequence
read from Chromas 1.45, Conor McCarthy
In big genome
sequencing projects, the entire process is automated, from
cloning and isolating the DNA to preparing the mixes and running
the sequencer. Computer software is used to align different
sets of sequences to generate one long tiling path
that covers the sequence from one end of the genome to the
next.
The Human Genome Project
sequenced the entire human genome, and completed it years
before the scheduled deadline. However, sequence alone is
of very little value, and it must be subjected to complex
computer analysis to identify genes and other features of
interest. Once such annotation is complete,
we will have a better understanding of what the genome represents.
Fungal genomes are also being sequenced,
and the Saccharomyces cerevisiae genome
database can be viewed by anyone who has access to an
internet ready PC.
Genomics has inspired the creation of
proteomics, metabolomics and other "-omics" sciences.
Proteomics looks at the total proteins of a species (proteome)
and the metabolomicists look at how an organism’s metabolism
works. |
|
