1 FISH Techniques, FISH Probes and Their Applications in Medicine and Biology - An Overview
15
oligonucleotides, either singly or in a cocktail, have been used to detect
high abundance RNA in cells or tissues. RNA probes are generally labeled
by in vitro transcription whereby the probe sequence is cloned into a vector containing RNA polymerase promoter sites, and probe molecules are
generated using RNA polymerase-mediated incorporation of labeled nucleotides.
When DNA probes are used for the detection of DNA targets within cells
or tissues, a denaturation step is required for a successful hybridization to
take place. This process can be achieved either by chemical means or by
heating above the melting temperature of the DNA double helices.
In practice a combination of the two approaches is used. When double
stranded DNA is heated to a temperature above its melting temperature
(which is determined by both its length and DNA sequence) the two
strands separate. Usually the melting temperature is too high and the
time of incubation too long for an efficient denaturation to take place
and, at the same time, preserve morphology. To accommodate for this,
the inclusion of organic solvents in the denaturation and hybridization
has proven efficient. Formamide is the most commonly used of these
and it functions by destabilizing the double stranded structure of DNA
at a given temperature thereby reducing the effective melting temperature.
The DNA probe and the DNA target molecules can either be denatured
separately or by co-denaturation. It has been argued that separate denaturation preserves morphology better, whereas co-denaturation reduces
the number of practical steps. For these reasons separate denaturation
steps are most often used in molecular cytogenetics and co-denaturation
most often used when tissue sections are analyzed.
In principle, riboprobes and oligonucleotide probes are single stranded
just like cellular RNA. However, denaturation improves the sensitivity
probably by removing secondary structures of RNA probe and target.
When the probe and target molecules are single-stranded, the two types of
molecules are brought together under conditions favoring hybridization.
This is achieved by ensuring a high probe concentration and lowering the
temperature of incubation.
When dealing with probes such as chromosomes, chromosome segments, YACs, BACs and PACs, where many copies of disperse repeats
are expected to be present, it is necessary to modify hybridization conditions to avoid cross hybridization to other chromosomal regions containing these repeated sequences. This is done by the so-called chromosomal
in situ suppression (CISS) hybridization (Hulten et al. 1991), where the
Ad 3: Denaturation
of probe
and sample
Ad 4: Hybridization
of probe to sample
15
oligonucleotides, either singly or in a cocktail, have been used to detect
high abundance RNA in cells or tissues. RNA probes are generally labeled
by in vitro transcription whereby the probe sequence is cloned into a vector containing RNA polymerase promoter sites, and probe molecules are
generated using RNA polymerase-mediated incorporation of labeled nucleotides.
When DNA probes are used for the detection of DNA targets within cells
or tissues, a denaturation step is required for a successful hybridization to
take place. This process can be achieved either by chemical means or by
heating above the melting temperature of the DNA double helices.
In practice a combination of the two approaches is used. When double
stranded DNA is heated to a temperature above its melting temperature
(which is determined by both its length and DNA sequence) the two
strands separate. Usually the melting temperature is too high and the
time of incubation too long for an efficient denaturation to take place
and, at the same time, preserve morphology. To accommodate for this,
the inclusion of organic solvents in the denaturation and hybridization
has proven efficient. Formamide is the most commonly used of these
and it functions by destabilizing the double stranded structure of DNA
at a given temperature thereby reducing the effective melting temperature.
The DNA probe and the DNA target molecules can either be denatured
separately or by co-denaturation. It has been argued that separate denaturation preserves morphology better, whereas co-denaturation reduces
the number of practical steps. For these reasons separate denaturation
steps are most often used in molecular cytogenetics and co-denaturation
most often used when tissue sections are analyzed.
In principle, riboprobes and oligonucleotide probes are single stranded
just like cellular RNA. However, denaturation improves the sensitivity
probably by removing secondary structures of RNA probe and target.
When the probe and target molecules are single-stranded, the two types of
molecules are brought together under conditions favoring hybridization.
This is achieved by ensuring a high probe concentration and lowering the
temperature of incubation.
When dealing with probes such as chromosomes, chromosome segments, YACs, BACs and PACs, where many copies of disperse repeats
are expected to be present, it is necessary to modify hybridization conditions to avoid cross hybridization to other chromosomal regions containing these repeated sequences. This is done by the so-called chromosomal
in situ suppression (CISS) hybridization (Hulten et al. 1991), where the
Ad 3: Denaturation
of probe
and sample
Ad 4: Hybridization
of probe to sample