398
ILSE CHUDOBA and GABRIELE SENGER
6. Add 30 J.d of hybridization mix and dissolve the pellet thoroughly.
Store at -20°C.
Hybridization mix - 2 g Dextran sulfate
- 7 ml deionized Formamide
- 1 ml20xSSC
- 2 ml dd H 2 0
- pH 7.0
- Store in 500 ~l aliquots at -20°C
Fluorescence in situ hybridization (FISH)
Microdissection of chromosomes and chromosomal regions can be carried out for two major purposes.
1. It provides the most elegant way for the characterization of chromosomal aberrations, e.g. the composition of marker and derivative chromosomes as well as the exact breakpoint of deletions and translocations.
In these cases, the chromosomes in question are isolated, or special regions of interest are excised. In a so called reverse FISH experiment,
the resulting DNA libraries are hybridized back to normal metaphase
spreads thus revealing their chromosomal origin.
2. By use of microdissection, specifically tailored partial painting
probes can be generated and applied in a forward FISH experiment in
order to characterize chromosomal aberrations.
Probe preparation
In general, 4 ~l of the unpurified PCR product after labeling, is largely
sufficient for one hybridization. This corresponds to about 200-300 ng
of DNA. Following the above mentioned purification procedure this
amount of labeled DNA is contained in approximately 6 ~l of the finally
obtained hybridization mixture. Small aliquots (20 ~l) ofCotl-DNA, each
sufficient for one single experiment, are lyophilized. The 6 ~l of the hybridization mix with the labeled DNA are added to one lyophilized aliquot
of Cotl-DNA and mixed thoroughly. If only one probe is hybridized, the
total volume is increased by adding 6 ~l of hybridization mix. In case a
second probe should be hybridized simultaneously, 6 ~l of another probe
ILSE CHUDOBA and GABRIELE SENGER
6. Add 30 J.d of hybridization mix and dissolve the pellet thoroughly.
Store at -20°C.
Hybridization mix - 2 g Dextran sulfate
- 7 ml deionized Formamide
- 1 ml20xSSC
- 2 ml dd H 2 0
- pH 7.0
- Store in 500 ~l aliquots at -20°C
Fluorescence in situ hybridization (FISH)
Microdissection of chromosomes and chromosomal regions can be carried out for two major purposes.
1. It provides the most elegant way for the characterization of chromosomal aberrations, e.g. the composition of marker and derivative chromosomes as well as the exact breakpoint of deletions and translocations.
In these cases, the chromosomes in question are isolated, or special regions of interest are excised. In a so called reverse FISH experiment,
the resulting DNA libraries are hybridized back to normal metaphase
spreads thus revealing their chromosomal origin.
2. By use of microdissection, specifically tailored partial painting
probes can be generated and applied in a forward FISH experiment in
order to characterize chromosomal aberrations.
Probe preparation
In general, 4 ~l of the unpurified PCR product after labeling, is largely
sufficient for one hybridization. This corresponds to about 200-300 ng
of DNA. Following the above mentioned purification procedure this
amount of labeled DNA is contained in approximately 6 ~l of the finally
obtained hybridization mixture. Small aliquots (20 ~l) ofCotl-DNA, each
sufficient for one single experiment, are lyophilized. The 6 ~l of the hybridization mix with the labeled DNA are added to one lyophilized aliquot
of Cotl-DNA and mixed thoroughly. If only one probe is hybridized, the
total volume is increased by adding 6 ~l of hybridization mix. In case a
second probe should be hybridized simultaneously, 6 ~l of another probe