14
EIGIL KJELDSEN and STEEN K0LVRAA
direct labeling offers the possibility for signal amplification, for example
by tyramide (Bobrow et al. 1989).
Labeling of probes is most commonly performed using enzymatic
methods (Table 3). DNA probes are conveniently labeled either by
nick-translation or randomly primed extension, both of which introduce
labeled nucleotides into the probe molecules. The labeling efficiencies,
however, are often far from 100%. An alternative labeling method is by
chemical cross-linking which gives close to 100% labeling efficiency (Craig
et al. 1997). Probe labeling can also be carried out using polymerase chain
reaction (PCR) based methods, with either direct incorporation of labeled
nucleotides or by using labeled primers. DOP-PCR is often used when
flow-sorted or microdissected chromosomes are being labeled.
Oligonucleotides can be labeled during synthesis where 5'-labeled nucleotides can be directly incorporated into the molecule. Enzymatic procedures can also be used for either 3'- or 5'-labeling. One advantage of 3'labeling is that multiple probe labels can be introduced to form a "tail"
thus increasing the sensitivity of detection. A variant of this type of labeling is the primed in situ labeling (PRINS) technique where the labeling of
the hybridized oligonucleotide is performed in situ, i.e. an unlabeled oligonucleotide is hybridized to its target DNA sequence in the sample and
the labeling is then performed in situ using conjugated nucleotides and a
thermostable polymerase (Koch et al. 1989).
For RNA detection (mRNA or viral RNA), RNA probes (riboprobes)
are often used because RNA-RNA hybrids are more stable than DNA-RNA
hybrids. Riboprobe vectors can be used to generate both sense and antisense probes to allow control of hybridization and the unbound probe can
be digested using RNase which do not digest double stranded RNA. Also
Table 3. Labeling strategies of probes
Genomic DNA for CGH
Flow sorted or microdissected
chromosomes or regions, YACs
Cosmids, BACs, PACs
Plasmids
Oligonucleotides
In situ labeling, chemical labeling
RNA probes
Nick-translation DOP-PCR
DOP-PCR
Nick-translation
Primed labeling
Nick-translation
Primed labeling
PNA
Chemical labeling during synthesis
Sense or antisense strand- 5'-label
EIGIL KJELDSEN and STEEN K0LVRAA
direct labeling offers the possibility for signal amplification, for example
by tyramide (Bobrow et al. 1989).
Labeling of probes is most commonly performed using enzymatic
methods (Table 3). DNA probes are conveniently labeled either by
nick-translation or randomly primed extension, both of which introduce
labeled nucleotides into the probe molecules. The labeling efficiencies,
however, are often far from 100%. An alternative labeling method is by
chemical cross-linking which gives close to 100% labeling efficiency (Craig
et al. 1997). Probe labeling can also be carried out using polymerase chain
reaction (PCR) based methods, with either direct incorporation of labeled
nucleotides or by using labeled primers. DOP-PCR is often used when
flow-sorted or microdissected chromosomes are being labeled.
Oligonucleotides can be labeled during synthesis where 5'-labeled nucleotides can be directly incorporated into the molecule. Enzymatic procedures can also be used for either 3'- or 5'-labeling. One advantage of 3'labeling is that multiple probe labels can be introduced to form a "tail"
thus increasing the sensitivity of detection. A variant of this type of labeling is the primed in situ labeling (PRINS) technique where the labeling of
the hybridized oligonucleotide is performed in situ, i.e. an unlabeled oligonucleotide is hybridized to its target DNA sequence in the sample and
the labeling is then performed in situ using conjugated nucleotides and a
thermostable polymerase (Koch et al. 1989).
For RNA detection (mRNA or viral RNA), RNA probes (riboprobes)
are often used because RNA-RNA hybrids are more stable than DNA-RNA
hybrids. Riboprobe vectors can be used to generate both sense and antisense probes to allow control of hybridization and the unbound probe can
be digested using RNase which do not digest double stranded RNA. Also
Table 3. Labeling strategies of probes
Genomic DNA for CGH
Flow sorted or microdissected
chromosomes or regions, YACs
Cosmids, BACs, PACs
Plasmids
Oligonucleotides
In situ labeling, chemical labeling
RNA probes
Nick-translation DOP-PCR
DOP-PCR
Nick-translation
Primed labeling
Nick-translation
Primed labeling
PNA
Chemical labeling during synthesis
Sense or antisense strand- 5'-label