Zusammenfassung der Ressource
DNA Profiling
- Short Tandem Repeats
- Found within
introns
(intragenic
regions) that
are non-coding
- Inherited in the same
way as any genes
within the coding
regions exons
(expressed regions)
- short DNA
sequences
repeated
many
times
- Occur at the same
locus on both
chromosomes of a
homologous pair
- the number of
repeats on each of
the homologous
chromosomes can
be different
- the number of
repeats at a locus
varies between
individuals
- Large amount
of variation in
the number of
repeats at each
locus
- Therefore 2
individuals are
highly unlikely
to have the
same
combination of
STR's
- Obtaining the
DNA
- Restriction Enzymes
- Endonucleases
means they cut
the DNA at sites
within a strand
of DNA
- Found
naturally in
bacteria where
they cut up
invading viral
DNA
- Only cut DNA
at specific base
sequences,
usually 4 or 6
base pairs long
- If restriction sites are
either side of an STR
sequence, that
fragment of DNA will
remain intact, but it
will cut away the rest
of the genome
- the
repeated
sequences
remain
intact
- The names of
the restriction
enzymes reflect
the bacteria
from which
they originate
- Eg, EcoR1
comes from
a strain of
Ecoli
- They cut a DNA
sample into
fragments only
where their
specific restriction
sequence occurs.
- Polymerase Chain
Reaction
- the sample is
placed in a
reaction tube with
DNA polymerase,
DNA primers and
nucleotides
- DNA primers are
short DNA
sequences
complementary to
the DNA adjacent
to the STR
- They're
marked with
fluorescent
tags
- the reaction
tube goes into
the PCR
thermal cycler
- Heated to
95degreesC
which
seperates
the double
stranded
DNA
- Cool to 55degreesC
which optimises the
binding of the primer to
the target DNA
sequence in the sample
- Heat to 70degreesC which is
the optimum temperature for
the heat stable DNA
polymerase. The polymerases
attach and nucleotides are
added, extending the DNA
from the primer
- After 2 cycles,
copies that
are just the
STR sequence
fragment are
produced
- As the cycle continues,
huge numbers of the
targeted DNA
fragments are
produced
- Electrophoresis
- Visualising the fragments
- Gel is quite fragile and
the DNA fragments
are double stranded
after electrophoresis
if restriction enzymes
have been used.
- Southern blotting is
used to transfer the
fragments to a
more resilient nylon
or nitrocellulose
membrane.
- The membrane is
placed directly
onto the gel and
a wad of dry
absorbent paper
placed on top.
- This acts as a wick to
draw buffer solution
up through the gel,
carrying DNA
fragments onto the
membrane.
- During this process,
the fragments
maintain their
positions relative to
each other and are
denatured into single
strands, exposing the
base sequences.
- The membrane is
then incubated
with an excess of
a labelled DNA
probe
- DNA probe: a short
section of DNA with
a base sequence
complementary to
the target DNA
sequence that needs
to be located
- After allowing time for
the probe to bind to
any complementary
sequences (hybridise),
any unbound probe is
washed away
- Probes may be
radioactive, labelled with
radioactive phosphorus,
or labelled with a
fluorescent marker
- With a radioactive
probe, the
membrane is dried
and placed next to
X-ray film.
- The film blackens
wherever the probe
has bound with the
DNA to form
double-stranded
fragments.
- If the probe is
fluorescent, its position
on the membrane can
be visualised under UV
light
- A single band occurs on
the profile where a
person's maternal and
paternal chromosomes
have the same number
of repeats at a particular
locus.
- 2 bands occur on the
profile if the 2
chromosomes have a
different number of
repeats at a locus
- There must be
a reference
profile for
comparison
- The reference profile may
come from a suspect in a
murder investigation, a
relative in the case of
identifying a corpse, or from
the parents when
establishing paternity
- The DNA
primers have a
fluorescent tag
attached to
them, which
allows the
system to be
automated
- As the DNA
fragments with their
attached fluorescent
tags move through
the gel they pass a
laser, the dye in the
tag fluoresces and
the coloured light is
detected
- This effectively
gives a time that it
has taken for the
fragments to pass
through the gel.
- Passing a separate set of
fragments of known
length through the gel
allows the length of time
for passage through the
gel to be calibrated with
fragment size
- Several STR loci can be
analysed at once by
using tags that
fluoresce at different
wavelengths giving
different colours for
each of the STR loci.
- A computer processes
the information from
the detector displaying
the the results of the gel
electrophoresis as a
graph.
- DNA fragments
are separated
according to
their size
- DNA placed on agarose
or polyacrymalide,
which provide a stable
medium through which
the fragments can
move (porous)
- Gel submerged in a
buffer solution, and
connected to
electrodes that
produce a potential
difference (voltage)
across the gel
- The negatively
charged DNA
fragments migrate
through the gel
according to their
overall charge and size
- In a given time,
smaller
fragments end
up closer to the
positive
electrode
- A reference sample
with fragments of
known length may
be added to the gel.
This is known as a
ladder or marker.
- The
fragments
are measured
in base pairs
- can come
from almost
all biological
tissue
- animal
or plant
- Eg, cells in
cheek swab,
WBC's in blood
smear, bone
marrow in a
skeleton, sperm
left after a
sexual assault
- Tissue sample
broken down in
buffer solution
(includes salt and
detergent) to
disrupt the cell
membranes
- the small suspended
particles, including
the DNA are
separated from the
rest of the cell debris
by filtering or
centrifuging
- Protease
enzymes are
incubated
with the
suspension to
remove
proteins
- Then, cold
ethanol is
added to
precipitate
out the DNA
- Several stages
of washing the
DNA in buffer
solution follow
- Inherited in the
same way as allelles
of a gene, with
offspring receiving
one repeated
sequence randomly
from each parent.
- Genetic profiling can be
used for identification
purposes, settling
paternity disputes,
identifying stolen
animals and looking at
variation and
evolutionary
relationships between
organisms
- Is DNA profiling infallible?
- Widely used in legal proceedings
- Generally thought to
produce a result that is
almost unique to the
individual and a near
certain indication of
guilt in criminal trials
- But because, the DNA
profile analyses only a
few repeated
sequences, it is less
likely to be completely
unique
- This is a particular
problem if
individuals being
tested are closely
related.
- In court, a forensic scientist
estimates the chance of seeing the
same DNA profile in the general
population; this is usually in excess of
1 billion
- (even for identical
twins, due to the
accumulation of
mutations during a
person's life)
- The same principles of
separating DNA fragments
are used when screening for
genetic conditions like cystic
fibrosis, measuring genetic
diversity or studying
evolutionary relationships