Probe

=Probe=

A **probe** is a custom-made oligonucleotide, either DNA or RNA, which is used to fish out a target nucleic acid in a mixture of fragments by hybridisation. Probes are labelled for detection, enabling the precise location of the target molecule.

There are many methods for incorporating a label into a probe, some of which are described below:


 * 1. Nick translation: ** dsDNA is randomly nicked using a low concentration of the enzyme DNase I. The nicks are then chewed away 5'-3' and filled in using a mixture of regular and labelled dNTPs. Both the 5'-3' exonuclease and polymerase activities are performed by DNA polymerase I. The labelled dNTPs should carry the label on their alpha phosphate ( 32 P) since this is the phosphate group which is incorporated into the growing DNA strand. The product of nick translation is a dsDNA probe with randomly labelled nucleotides along its length.


 * 2. Primer extension using random hexamers: ** denatured (single-stranded) probe DNA is incubated with random hexamers, such as GACCTG, some of which will randomly anneal to it. A Klenow fragment (DNA polymerase that lacks 5'-3' exonuclease activity) is then used to polymerise the gaps between the annealed hexamer primers. Again, labelled or chemically modified dNTPs are used in polymerisation. It is crucial that DNA polymerase lacks 5'-3' exonuclease activity because otherwise hexamer primers in front of the path of polymerisation will be chewed away. The product is, again, a dsDNA probe with random regions of labelled nucleotides.


 * 3. Primer extension using specific oligonucleotide primers: ** uses the same procedure as above, except with a single, longer and sequence-specific primer. This can only be used if you know the sequence of the DNA being used as a probe.


 * 4. Riboprobes: ** this procedure involves cloning the probe into a plasmid, downstream of a promoter than can be recognised by a phage-encoded RNA polymerase. The plasmid is then linearised with restriction enzymes and transcribed (using labelled NTPs) up to the end of the plasmid template ('run-off transcription'). This produces a cluster of labelled RNA probes - //riboprobes//. Riboprobes can also be synthesised in a PCR fashion:


 * Template DNA is denatured to single-stranded forfm
 * A PCR primer is attached to ssDNA with the phage promoter sequence attached to its 5' end
 * The primer is extended (from its 3' end) to make a new template strand
 * Another PCR primer is attached to the new template strand; when this is extended, the phage promoter sequence will also be incorporated
 * The product is a DNA containing the phage promoter, which can then be transcribed with labelled NTPs as above


 * 5. End-labelling with a single label: ** this involves first using alkaline phosphatase, to remove the 5' phosphate of the prospective probe, and then using polynucleotide kinase and radio-labelled ATP (on the //gamma// phosphate) to incorporate a radio-labelled 5' phosphate in its place. When labelling oligonucleotides in this way, the phosphatase step can be omitted because oligonucleotides lack a 5' phosphate. This is not so useful where longer DNA molecules are used as probes, however, because only one label is incorporated per strand.


 * 6. End-labelling with multiple labels: ** each strand of a dsDNA is exonucleolytically digested in a 3'-5' fashion using polymerase and then multiple labelled dNTPs are incorporated in place of the digested regions.

The label used may either be radioactive or non-radioactive. Radioactive labels are usually incorporated in the form of 32 P, in either alpha-dNTP or gamma-NTP. Non-radioactive labels include biotin or deoxygenin. Both of these are incoporated as modified UTP or dUTP; the modified deoxy-uracil residues can themselves be probed for using fluorescently-tagged or enzyme-conjugated antibodies.


 * The incorporation of biotin is called //biotinylation//. The DNA probe has biotin-dUTP incorporated into it either by nick translation, random priming or end-filling (as described above) and the biotinylated DNA probe is then hybridised to the target. The probe is detected by 'probing for the probe' with avidin, which itself is either coupled to a fluorescent marker or conjugated with an enzyme such as horseradish peroxidase (the peroxidase catalyses a bioluminescent reaction when provided with its substrate; the bioluminescence thus acts as the detection marker)


 * The incorporation of deoxygenin is via deoxygenin-dUTP, incorporated into the DNA probe via nick translation, random priming or end-filling, as above. Once the probe has bound to its target, the probe is 'probed for' using an anti-DIG antibody. The antibody may either be coupled to a fluorescent tag or an enzyme conjugate - again, such as horseradish peroxidase.