1 FISH Techniques, FISH Probes and Their Applications in Medicine and Biology - An Overview
13
For the detection of whole chromosomes (Pinkel et al. 1986, Lichter et al.
1988, Pinkel et al. 1988, Hulten et al. 1991) or a part thereof (from individual bands (Guan et al. 1993) to arms (Guan et al. 1996» libraries are
generally generated from normal individuals or cell hybrids containing
human chromosomes. By fluorescence activated chromosome sorting
of metaphases from these sources, chromosome specific libraries for
each human chromosome, 1-22, X and Y, have been generated (Oeaven
et al. 1986, Carter et al. 1992). A sorting experiment can, in general, generate approximately 500 copies of each chromosome. These can then be
amplified by OOP-PCR in which a degenerate primer is used in a special
PCR-amplification step as described by Telenius et al. (1992). The composite nature of these probe mixtures again ensures that a sufficient number of fluorochromes are bound to the region of interest. Another way of
isolating whole chromosomes or parts thereof is by microdissection (Guan
et al. 1993, Thangavelu et al. 1994). This can be done by micromanipulators (Meltzer et al. 1992) or laser-equipped devices (Monajembashi
et al. 1986). Here only 25-50 copies of each chromosome can be isolated
and further amplified by OOP-PCR before fluorescent labeling. Even
a single microdissected chromosome can be amplified and used as a
chromosome painting probe (Christian et al. 1999, Kjeldsen unpublished
data).
The choice of label is governed to some extent by personal preference
and, in some situations, by commercial availability. Although isotopic
(radioactive) labeling initially was the choice of label because of its sensitivity, it is now largely confined to RNA detection, particularly when
non-isotopic measures have been unsuccessful. Radioactive sulfur has
been the most frequently used label as it combines specific activity
with relatively high morphological resolution.
Non-isotopic ISH sensitivity has been greatly improved and most laboratories are now using non-isotopic labels exclusively. The most commonly used non-isotopic labels are fluorescent dyes such as FITC, rhodamine and Texas Red, which can be coupled to nudeotides, antibodies or
macromolecules. There are two basic methods for non-isotopic labeling:
(1) fluorescent - direct or indirect; and (2) non-fluorescent. The probes
are labeled either directly with nucleotides coupled to a fluorochrome or
indirectly with nucleotides coupled to a reporter-molecule, which subsequently can be detected by conventional immunochemical methods. The
most commonly used indirect labels are biotin and digoxigenin coupled to
nucleotides which - after incorporation - can be detected using fluorescence-conjugated avidin or antibodies, respectively. The choice of direct
or indirect labeling is mainly determined by demand for sensitivity as in-
13
For the detection of whole chromosomes (Pinkel et al. 1986, Lichter et al.
1988, Pinkel et al. 1988, Hulten et al. 1991) or a part thereof (from individual bands (Guan et al. 1993) to arms (Guan et al. 1996» libraries are
generally generated from normal individuals or cell hybrids containing
human chromosomes. By fluorescence activated chromosome sorting
of metaphases from these sources, chromosome specific libraries for
each human chromosome, 1-22, X and Y, have been generated (Oeaven
et al. 1986, Carter et al. 1992). A sorting experiment can, in general, generate approximately 500 copies of each chromosome. These can then be
amplified by OOP-PCR in which a degenerate primer is used in a special
PCR-amplification step as described by Telenius et al. (1992). The composite nature of these probe mixtures again ensures that a sufficient number of fluorochromes are bound to the region of interest. Another way of
isolating whole chromosomes or parts thereof is by microdissection (Guan
et al. 1993, Thangavelu et al. 1994). This can be done by micromanipulators (Meltzer et al. 1992) or laser-equipped devices (Monajembashi
et al. 1986). Here only 25-50 copies of each chromosome can be isolated
and further amplified by OOP-PCR before fluorescent labeling. Even
a single microdissected chromosome can be amplified and used as a
chromosome painting probe (Christian et al. 1999, Kjeldsen unpublished
data).
The choice of label is governed to some extent by personal preference
and, in some situations, by commercial availability. Although isotopic
(radioactive) labeling initially was the choice of label because of its sensitivity, it is now largely confined to RNA detection, particularly when
non-isotopic measures have been unsuccessful. Radioactive sulfur has
been the most frequently used label as it combines specific activity
with relatively high morphological resolution.
Non-isotopic ISH sensitivity has been greatly improved and most laboratories are now using non-isotopic labels exclusively. The most commonly used non-isotopic labels are fluorescent dyes such as FITC, rhodamine and Texas Red, which can be coupled to nudeotides, antibodies or
macromolecules. There are two basic methods for non-isotopic labeling:
(1) fluorescent - direct or indirect; and (2) non-fluorescent. The probes
are labeled either directly with nucleotides coupled to a fluorochrome or
indirectly with nucleotides coupled to a reporter-molecule, which subsequently can be detected by conventional immunochemical methods. The
most commonly used indirect labels are biotin and digoxigenin coupled to
nucleotides which - after incorporation - can be detected using fluorescence-conjugated avidin or antibodies, respectively. The choice of direct
or indirect labeling is mainly determined by demand for sensitivity as in-