2. NUCLEOTIDE PROBES
A probe is defined as a single stranded piece of
DNA, labelled (either with radioisotope or with
non-radioactive label), the nucleotide sequence of
which is complementary to the target DNA.
It is a single stranded piece of DNA (sometimes
RNA), which can range in size from as little as 15
bp to several hundread kilobases.
It can identify, through base pairing, a specific
DNA fragment of the library, which contains
complementary sequence.
3. Probe Type Size Origin
DNA 0.1 – 100kb Cell-based DNA
Cloning, PCR
RNA (or Riboprobe) 1 – 2 kb RNA transcription
from plasmid (or
phage vectors)
Oligonucleotide 15 – 50 nucleotides Chemical Synthesis
4. A probe DNA will form complementary base
pairing with another DNA strand.
mRNA can be used as a probe: it will bind to the
DNA fragment that contains exon sequences of
it’s gene.
RNA probe, termed riboprobe, can be produced
by in vitro transcription of cloned DNA inserted
into a plasmid vector.
Synthetic oligonucleotide probes; constructed by
chemical methods, are most commonly used.
5. PROBES MUST HAVE A LABEL TO BE
IDENTIFIED
Probes are labelled with the radioisotopes, such as
32
p or tritium.
These probes can be detected by autoradiography,
which involves placing the sample in direct contact
with the photographic material, usually X-ray film.
Alternatively, end-labelling probes with fluorescent
tags can be used. The latter are visible under the
UV-Lamp.
6. TECHNIQUES FOR LABELLING
PROBES
There are two general ways in which a labelled
nucleotide can be incorporated into the structure of
the probe:
1) End-Labelling:
Addition of a labelled group to one terminal of the
probe is done.
For eg:- By exchanging a labelled γ-Phosphate from
ATP with a phosphate from the 5’- terminal on (single
or double- stranded) DNA.
7. 2) Polymerase – based Labelling:-
Using a DNA Polymerase, multiple- labelled –
nucleotides are incorporated into the probe
during DNA synthesis.
Such a reaction requires dNTPs & it is
customary to have one of them to be labelled.
Eg: dGTP
Because on an average 25% of the nucleotides
incorporated are labelled.
This type has a higher specific activity than the
end-labelling where only terminal nucleotide is
labelled.
8. USES OF NUCLEOTIDE PROBES:
i. To search specific DNA sequences of DNA library,
as discussed.
ii. In southern & Northern blot techniques, probes
are used to identify DNA or RNA fragments
respectively.
iii. In diagnosis of genetic disorders, such as sickle
cell anaemia, thalassaemia, cystic fibrosis etc.
NOTE:- Probe gets involved in the formation of heteroduplex with
template DNA, and this is the key to usefulness of molecular
hybridization.
