1 FISH Techniques, FISH Probes and Their Applications in Medicine and Biology - An Overview
25
Padlock probes
A strategy to improve discrimination of in situ hybridization of single base
mismatches is the so-called padlock probes (Nilsson et al. 1994). These
probes are oligonucleotides with probe sequences of about 20 nucleotides
on the 3'- and 5'-end with a 30- to 40-mer spacer in between, to which
haptens (e.g. biotin and digoxigenine) are coupled for detection purposes.
The probe sequences are chosen such that in a hybrid molecule, the 3'- and
5'-ends of the padlock probe are juxtaposed and can then be covalently
linked by a ligation step to close the padlock probe. This setup provides
three important features with respect to uniqueness ofhybridization. First,
the formation of a circular molecule allows for extreme stringency
conditions in the washing step, secondly, the juxtaposition of the probe
ends requires two independent hybridization events, and thirdly the DNA
ligase used for ligation is highly dependent on perfect match. The padlock
methodology has successfully been used to detect single base mismatches
of repeat sequences by fluorescence in situ hybridization on metaphase
spreads (Nilsson et al. 1997). However, sensitivity is still an issue but signal
amplification by a rolling circle mechanism is a possible way to achieve the
sensitivity for single copy sequences (Daubendiek and Kool 1997).
Applications of FISH in medicine and biology
General considerations (Table 4)
The most powerful and important feature of FISH is that the technique
allows detection of a particular gene or genetic sequence at the subcellular
level in a multi-color mode. In basic science FISH has strongly enhanced
gene and genome analysis, enabling a rapid mapping of genes. Diagnostically FISH allows detection of abnormalities that otherwise could not
have been identified or only with great difficulty, including identification
of pathological chromosome abnormalities in oncology, congenital malformations and single gene disorders. FISH techniques offer the possibility
of selecting where in the genome staining shall occur and the technique is
therefore used either to complement conventional banding methods or as
a substitute for chromosome identification at metaphase or interphase.
FISH also has the advantage of being able to detect microbial DNA or
RNA sequences without the need for prior propagation of cells or microorganisms.
25
Padlock probes
A strategy to improve discrimination of in situ hybridization of single base
mismatches is the so-called padlock probes (Nilsson et al. 1994). These
probes are oligonucleotides with probe sequences of about 20 nucleotides
on the 3'- and 5'-end with a 30- to 40-mer spacer in between, to which
haptens (e.g. biotin and digoxigenine) are coupled for detection purposes.
The probe sequences are chosen such that in a hybrid molecule, the 3'- and
5'-ends of the padlock probe are juxtaposed and can then be covalently
linked by a ligation step to close the padlock probe. This setup provides
three important features with respect to uniqueness ofhybridization. First,
the formation of a circular molecule allows for extreme stringency
conditions in the washing step, secondly, the juxtaposition of the probe
ends requires two independent hybridization events, and thirdly the DNA
ligase used for ligation is highly dependent on perfect match. The padlock
methodology has successfully been used to detect single base mismatches
of repeat sequences by fluorescence in situ hybridization on metaphase
spreads (Nilsson et al. 1997). However, sensitivity is still an issue but signal
amplification by a rolling circle mechanism is a possible way to achieve the
sensitivity for single copy sequences (Daubendiek and Kool 1997).
Applications of FISH in medicine and biology
General considerations (Table 4)
The most powerful and important feature of FISH is that the technique
allows detection of a particular gene or genetic sequence at the subcellular
level in a multi-color mode. In basic science FISH has strongly enhanced
gene and genome analysis, enabling a rapid mapping of genes. Diagnostically FISH allows detection of abnormalities that otherwise could not
have been identified or only with great difficulty, including identification
of pathological chromosome abnormalities in oncology, congenital malformations and single gene disorders. FISH techniques offer the possibility
of selecting where in the genome staining shall occur and the technique is
therefore used either to complement conventional banding methods or as
a substitute for chromosome identification at metaphase or interphase.
FISH also has the advantage of being able to detect microbial DNA or
RNA sequences without the need for prior propagation of cells or microorganisms.