314
Anthrax (Bacillus anthracis), Molecular Biology of
3. Amplify fragments by polymerase chain reaction using labeled primers complementary to the linker sequences
2. Ligate restriction fragments to linker sequences
1. Cut genome with two restriction enzymes
4. Separate fragments by size using gel electrophoresis
Fig. 3
The basic method for AFLP analysis. In
this method, DNA from a sample is cut into
pieces using restriction enzymes. The size of
these fragments should differ between samples,
based on the location of restriction enzyme sites
in the DNA sequences. Rather than amplifying
the DNA restriction fragments using sequences
within them, linkers of known DNA sequence are
attached to the ends of these fragments so that
they may be ampli±ed using primers
complementary to the linker fragments. This
allows one to amplify DNA even when the
sequences in the target organism are unknown.
The ampli±ed fragments can then be separated
by size so that the patterns of fragments
between samples can be compared.
genetic makeup of the target organism
is
necessary.
The
AFLP
pro±le
of
a
sampled organism can be compared to
that of known pro±les for identi±cation.
T
h
e
s
ec
om
p
a
r
i
s
o
n
sc
a
nb
el
o
g
i
s
t
i
c
a
l
l
y
dif±cult, especially when a large number
of fragments are examined at once. To
make
this
easier,
AFLP
pro±les
for
a
number of organisms are being archived
and
algorithms
developed
to
automate
these
comparisons.
This
method
does
show promise for use in the identi±cation
of
B. anthracis
, because the AFLP pro±les
generated
for
a
variety
of
B. anthracis
strains show signi±cant differences from
closely related
Bacillus
species.
Long-range repetitive element-polymor-
phism
PCR
(LR
REP-PCR)
is
another
method that uses length polymorphisms
to distinguish between bacterial species.
This
technique
uses
highly
conserved
repetitive extragenic palindromic (REP)
sequences in DNA as target sequences
in a PCR reaction. DNA sequences that
lie between two adjacent REP sequences
are ampli±ed in these reactions (Fig. 4).
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