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Probes -
Probes
are
small
(15-30
bases
long)
nucleotide
sequences
used
to
detect
the
presence
of
complementary
sequences
in
nucleic
acid
samples.
This
is
achieved
by
permitting
the
probes
to
base
pair
with
the
sample
nucleic
acids
and
then
identifying
the
samples
that
show
base
pairing
with
the
probes,
i.e.,
hybridization.
The
detection
of
hybridization
is
highly
precise
and
extremely
sensitive
provided
the
probes
are
suitably
labelled
for
an
easy
detection.
Clearly
hybridization
can
occur
only
when
the
base
sequence
of
a
probe
is
present
within
the
gene
or
DNA
segment,
which
it
is
aimed
to
detect.
Both
DNA
and
RNA
are
used
as
probes.
Single
stranded
DNA
probes
are
more
convenient
and
preferable,
but
denatured
double
stranded
DNA
molecules
can
also
be
used.
RNA
probes
are
ordinarily
single
stranded.
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Preparation
of
Probes Probes
can
be
obtained
in
several
ways;
some
of
the
important
ones
are
briefly
described
below.
1.
Highly
purified
mRNA
can
be
used
as
probe;
mRNA's
are
naturally
single
stranded.
2.
Single
strand
RNA
probes
can
be
readily
prepared
by
cloning
the
corresponding
DNA
sequence
inserted
into
a
special
vector
like
pGEM
or
phage
M13
vectors.
pGEM
has
a
different
and
specific
prokaryotic
promoter
on
the
two
ends
of
the
DNA
insert.
The
recombinant
DNA
is
linearized
and
transcribed
in
vitro
with
the
appropriate
prokaryotic
RNA
polymerase
to
obtain
RNA
molecules
complementary
to
one
or
the
other
strand
of
the
DNA
insert.
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3.
DNA
segments
isolated
from
the
genome
of
an
organism
or
cDNA
molecules
prepared
by
using
mRNAs
can
be
cloned
in
E.
coli
and
used
as
probes.
These
probes,
of
necessity,
will
be
double
stranded.
4.
Single
stranded
cDNA
probes
can
be
prepared
by
limiting
the
copying
of
mRNA
by
reverse
transcriptase
to
only
one
strand.
5.
PCR
can
be
used
to
generate
single
stranded
copies
of
a
DNA
sequence
by
asymmetric
PCR.
6.
Synthetic
oligonucleotides
can
be
prepared
for
use
as
probes.
This
is
rather
easy
when
the
base
sequence
of
DNA
to
be
detected
or
of
RNA
produced
by
it
is
known.
But
the
base
sequence
of
a
part
of
the
gene
can
be
deducted
from
the
amino
acid
sequence
of
a
small
segment,
say,
5
amino
acid
long
of
the
protein
encoded
by
it.
But
it
is
impossible
to
determine
the
exact
base
sequence
of
the
gene
due
to
the
degeneracy
of
genetic
code,
i.e.,
one
amino
acid
encoded
by
two
or
more
codons.
This
problem
is
tackled
by
synthesizing
a
mixture
of
oligonucleotides
containing
all
the
possible
base
sequences
predicted
by
the
degenerate
code.
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For
example,
there
are
four
different
base
sequences
possible
on
the
basis
of
the
amino
acid
sequence.
The
oligonucleotide
mixture
should
contain
all
the
four
sequences.
Labelling
of
Probes.
The
probes
can
either
be
labelled
with
(1)
radioactivity,
e.g.,
32p,
or
(2)
with
nonradioactive
labels,
e.g
biotin,
digoxigenin,
etc.
1. Radioactive
Labelling.
The
various
techniques
for
labelling
of
nucleic
acids
are
as
follows:
(1)
nick
translation
(Appendix-2.IX),
(2)
primer
extension,
(3)
methods
based
on
RNA
polymerase,
(4)
end
labelling
(see,
Appendix2.IV),
and
(5)
direct
labelling
methods.
In
direct
labelling
a
nucleotide
labelled
with
32p
is
provided
during
production
of
the
probe
so
that
this
radioactive
nucleotide
is
included
in
the
probe.
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2.
Nonradioactive
Labelling.
There
are
several
strategies
for
nonradioactive
labelling
of
nucleic
acids,
e.g.,
labelling
with
biotin,
digoxigenin,
fluorescent
molecules,
etc.
Radioactive
labelling
poses
problems
in
handling
and,
especially,
disposal.
Further
due
to
the
relatively
short
half
life
of
the
radioisotopes
the
probes
have
to
be
used
in
a
short
period
of
time
after
they
are
prepared.
In
contrast,
some
nonradioactively
labelled
probes
can
be
stored
at
-20°C
for
long
periods
of
time.
Biotin
labelled
probes
are
prepared
by
nick
translation
in
which
biotin
conjugated
(biotinylated)
nucleotides
are
used.
Long
tails
of
biotinylated
nucleotides
may
be
added
to
the
probes
to
increase
the
number
of
biotin(vit.
H)
molecules
available
for
the
colour
development
necessary
for
detection;
these
tails
have
a
nonspecific
base
sequence
so
that
the
same
tail
can
be
added
to
any
probe.
These
probes
are
hybridized
with
the
test
nucleic
acid
fixed
to
a
solid
support
and
the
nonhybridized
probes
are
washed
off.
The
hybridized
samples
are
detected
by
the
development
of
blue
colour
following
a
series
of
cytochemical
reactions,
which
basically
utilize
the
affinity
of
egg
white
glycoprotein
avidin
for
biotin.
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This
approach
has
the
advantage
of
being
much
faster
than
that
with
radioactive
probes,
which
requires
autoradiography.
But
its
chief
disadvantage
relates
to
the
inability
to
reuse
the
filter
(solid
support)
and
the
nucleic
acids
fixed
to
it
for
hybridization
with
other
probes
since
the
reactions
leading
to
the
colour
development
produce
insoluble
precipitates.
In
contrast,
the
radioactive
probes
are
easily
removed
by
washing
under
conditions
favouring
denaturation,
e.g.,
high
pH,
and
the
same
filter
can
be
reused
for
hybridization
with
a
series
of
probes,
used
one
at
a
time.
Nucleotides
conjugated
with
digoxigenin,
a
plant
derived
chemical,
may
be
used
in
nick
translation
to
produce
digoxigenin
labelled
probes.
The
probes
are
used
in
hybridization;
after
washing
away
the
free
probes,
the
filter
is
incubated
in
a
detection
buffer
containing
a
digoxigenin
specific
antibody
(anti
digoxigenin)
coupled
with
an
enzyme
(usually
alkaline
phosphatase).
After
appropriate
washing,
the
alkaline
phosphatase
activity
is
detected
by
using
a
suitable
substrate
that
yields
colour
due
to
the
enzyme
action.
Nucleotides
have
been
conjugated
with
other
ligands
that
produce
some
detectable
signal,
e.g.,
fluorescence,
enzyme
activity,
etc.
The
signals
from
probes
can
be
amplified
by
anyone
of
several
approaches,
e.g.,
(i)
attaching
multiple
enzyme
molecules
to
each
probe
molecule,
(ii)
adding
nonspecific
labelled
tails
to
the
probes.
(iii)
Using
multiple
secondary
probes
that
hybridize
with
multiple
target
specific
primary
probes
(Christmas
tree
or
forest
approach),
etc.
Applications
of
Probes
1.
Identification
of
the
recombinant
clones
carrying
the
desired
DNA
inserts;
this
is
the
most
critical
step
in
DNA
cloning
(techniques used:
Southern,
northern,
colony,
dot
blot
hybridizations).
2.
Confirmation
of
the
integration
and
detection
of
copy
number
(by
Southern
hybridization)
of
a
DNA
insert
in
the
host
genome,
and
its
expression
in
transformed
cells
(northern
hybridization).
3.
Development
of
RFLP
(restriction
fragment
length
polymorphism)
maps.
4.
DNA
finger
printing
for
the
unequivocal
identification
of
plant
varieties,
criminals,
parental
relationships,
etc.
5.
In
situ
hybridization
(Appendix-2.X)
for
determining
the
locations
of
specific
sequences
in
specific
chromosomes.
6.
Accurate
diagnosis
of
diseases
caused
by
parasites,
pathogens
or
defective
viruses.
7.
Preparation
of
genome
maps
of
eukaryotes,
including
man.
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