372
JINGLY FUNG et al.
Subprotocol 2
Preparation of probes and hybridization mixture
Caution: The preparation of probes is best performed with kits which can
be purchased from anyone of several major companies (such as Roche
Molecular Biochemicals, Life Technologies, VYSIS, Applied Spectral Imaging, Inc. and others). The DNA should be free of contaminants which
might inhibit DNA polymerases, such as metals, detergents etc. In
many cases, an alkaline lysis DNA isolation protocol followed by phenol-chloroform extraction and isopropanol precipitation should be sufficient. The PFGE agarose gel purification for YAC clones described below,
as well as most cesium chloride gradient DNA isolation protocols will also
purify DNA sufficiently for random priming.
Procedure
Whole chromosome painting probes for metaphase analysis
The chromosome painting probe for metaphase cell analysis is generated
by flow sorting of human chromosomes and DNA amplification using degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius 1992). The
chromosome specific PCR products are labeled by directly incorporating
haptenized (biotin and digoxigenin) or fluorochrome (Spectrum Green,
Spectrum Orange, Texas Red) conjugated nucleotides, incorporated by
PCR (Telenius 1992). Probes labeled with biotin or digoxigenin are detected with avidin-Cy5 and Cy5.5-conjugated antibodies against digoxin.
Kits including all 24 chromosomes painting probes (viall of SKY kit) can
be purchased from Applied Spectral Imaging, Inc.
locus-specific probes for interphase analysis: A. Preparation of DNA from yeast
artificial chromosome (YAe)
The DNAs from YACs are isolated using pulsed field gel electrophoresis
(PFGE) and amplified by degenerate oligonucleotide-primed PCR (DOPPCR).
1. Grow the selected yeast clone (containing the YAC) on ARC agar for
2-3 days at 30°e. Pick colonies from the plates and culture the colonies
in 5-35 ml AHC media at 30°C for 2-3 days in a shaking incubator.
JINGLY FUNG et al.
Subprotocol 2
Preparation of probes and hybridization mixture
Caution: The preparation of probes is best performed with kits which can
be purchased from anyone of several major companies (such as Roche
Molecular Biochemicals, Life Technologies, VYSIS, Applied Spectral Imaging, Inc. and others). The DNA should be free of contaminants which
might inhibit DNA polymerases, such as metals, detergents etc. In
many cases, an alkaline lysis DNA isolation protocol followed by phenol-chloroform extraction and isopropanol precipitation should be sufficient. The PFGE agarose gel purification for YAC clones described below,
as well as most cesium chloride gradient DNA isolation protocols will also
purify DNA sufficiently for random priming.
Procedure
Whole chromosome painting probes for metaphase analysis
The chromosome painting probe for metaphase cell analysis is generated
by flow sorting of human chromosomes and DNA amplification using degenerate oligonucleotide primed PCR (DOP-PCR) (Telenius 1992). The
chromosome specific PCR products are labeled by directly incorporating
haptenized (biotin and digoxigenin) or fluorochrome (Spectrum Green,
Spectrum Orange, Texas Red) conjugated nucleotides, incorporated by
PCR (Telenius 1992). Probes labeled with biotin or digoxigenin are detected with avidin-Cy5 and Cy5.5-conjugated antibodies against digoxin.
Kits including all 24 chromosomes painting probes (viall of SKY kit) can
be purchased from Applied Spectral Imaging, Inc.
locus-specific probes for interphase analysis: A. Preparation of DNA from yeast
artificial chromosome (YAe)
The DNAs from YACs are isolated using pulsed field gel electrophoresis
(PFGE) and amplified by degenerate oligonucleotide-primed PCR (DOPPCR).
1. Grow the selected yeast clone (containing the YAC) on ARC agar for
2-3 days at 30°e. Pick colonies from the plates and culture the colonies
in 5-35 ml AHC media at 30°C for 2-3 days in a shaking incubator.