1 FISH Techniques, FISH Probes and Their Applications in Medicine and Biology - An Overview
II
A crucial point when deciding on the choice of probe type and label is
sensitivity, Le. one must consider how the target sequence of interest can
be detected. This depends on a several factors: (1) size of target sequence
(large genes/nucleic acid sequences can bind more probe and hence more
label); (2) is the target sequence unique or does it exist in multiple copies
(from a few to several thousand copies) at the site oflocation (the more
copies, the more label can be bound); (3) size of probe sequence (theoretically, large probes can carry more label but the maximum probe size in
the hybridization mixture is 300 to 500 base pairs as larger probe fragments either have difficulty in penetrating the target sequence or form
large aggregates which reduces the amount of available probe and increases background); and 4) indirect labeling techniques offer the possibility for signal amplification.
In Table 2 the different probe types are listed with respect to size of
probe sequences, which stretch from very short synthetic oligonucleotides
up to composite probes representing a whole chromosome.
The short oligonucleotides (20 bases up to 100 bases) are synthetic
DNA, which can be labeled during manufacturing, but because of their
size, these sequences can carry only a few fluorochromes. This represents
a problem in sensitivity for the detection of unique sequences. However,
when dealing with short sequences that are moderately to highly repeated,
such as centromeres and telomers, these short probes are very efficient
(Koch et al. 1989, Gosden et al. 1991, Hindkjrer et al. 1994, Therkelsen
et al. 1995).
For detection of single genes or gene regions, cloned probes are often
used (Kievits et al. 1990, Rowley et al. 1990, Kuwano et al. 1991). The choice
of vector determines the size of the fragment that can be cloned. Cloned
DNA fragments in plasmids, cosmids, YACs (yeast artificial chromosomes), PACs (PI filamentous phage artificial chromosomes) or BACs
(bacterial artificial chromosomes) are the most commonly used probes.
These are named locus specific probes as the cloned fragment is derived
from a specific region of the metaphase.
Often other vectors than plasmids are preferred because of the huge
fragments of DNA that can be contained in them. This enables the specific
binding of numerous fluorochrome molecules to the region of interest
which, in principle, makes detection unproblematic. However, the cloned
fragments also contain families of interspersed repeated sequences which
at the same time are scattered throughout the genome. Therefore, methods
had to be developed to avoid or minimize the possibility of detecting these
repeated sequences at other "unspecific" sites in the metaphase (see below).
II
A crucial point when deciding on the choice of probe type and label is
sensitivity, Le. one must consider how the target sequence of interest can
be detected. This depends on a several factors: (1) size of target sequence
(large genes/nucleic acid sequences can bind more probe and hence more
label); (2) is the target sequence unique or does it exist in multiple copies
(from a few to several thousand copies) at the site oflocation (the more
copies, the more label can be bound); (3) size of probe sequence (theoretically, large probes can carry more label but the maximum probe size in
the hybridization mixture is 300 to 500 base pairs as larger probe fragments either have difficulty in penetrating the target sequence or form
large aggregates which reduces the amount of available probe and increases background); and 4) indirect labeling techniques offer the possibility for signal amplification.
In Table 2 the different probe types are listed with respect to size of
probe sequences, which stretch from very short synthetic oligonucleotides
up to composite probes representing a whole chromosome.
The short oligonucleotides (20 bases up to 100 bases) are synthetic
DNA, which can be labeled during manufacturing, but because of their
size, these sequences can carry only a few fluorochromes. This represents
a problem in sensitivity for the detection of unique sequences. However,
when dealing with short sequences that are moderately to highly repeated,
such as centromeres and telomers, these short probes are very efficient
(Koch et al. 1989, Gosden et al. 1991, Hindkjrer et al. 1994, Therkelsen
et al. 1995).
For detection of single genes or gene regions, cloned probes are often
used (Kievits et al. 1990, Rowley et al. 1990, Kuwano et al. 1991). The choice
of vector determines the size of the fragment that can be cloned. Cloned
DNA fragments in plasmids, cosmids, YACs (yeast artificial chromosomes), PACs (PI filamentous phage artificial chromosomes) or BACs
(bacterial artificial chromosomes) are the most commonly used probes.
These are named locus specific probes as the cloned fragment is derived
from a specific region of the metaphase.
Often other vectors than plasmids are preferred because of the huge
fragments of DNA that can be contained in them. This enables the specific
binding of numerous fluorochrome molecules to the region of interest
which, in principle, makes detection unproblematic. However, the cloned
fragments also contain families of interspersed repeated sequences which
at the same time are scattered throughout the genome. Therefore, methods
had to be developed to avoid or minimize the possibility of detecting these
repeated sequences at other "unspecific" sites in the metaphase (see below).