24 Fluorescence in Situ Hybridisation of a Complete Set of 41 Telomere Specific Probes
303
opposed to 48 (the p-arms of the acrocentric chromosomes are excluded
because they contain repetitive and ribosomal DNA only and the X and Y
chromosomes share homologous sequences at both pseudoautosomal regions). These probes were produced as cosmid clones with size ranges
from 20-40 kb, making the direct labelling of these for rapid FISH analysis
difficult. In addition, this first generation set contained probes which were
not as close to the end of the chromosome as would be preferred. More
recently, a second generation of probes have been developed. These
probes have been developed as PAC clones and are approximately 100
kb in size making the direct labelling of them possible. These new probes
are of varying distance from the telomere, ranging from 30-450 kb as a
result of the variable length of the TAR.
Standard FISH techniques would require the individual testing of every
telomere probe for each different chromosome, making routine screening
impractical. We have combined the complete set of specific telomere
probes with our Chromoprobe Multiprobe technology to produce a novel
FISH technique to allow the simultaneous hybridisation and analysis of all
probes on one slide. The Multiprobe is a disposable glass device with 24
raised bosses to which each of the 41 telomere probes are reversibly applied following a patented process. Each square thus contains its p-arm
probe in green and its q arm probe in red (except the acrocentric chromosomes which have q- arms only, and the sex chromosomes which share
their telomere specific sequences). This offers cytogenetic diagnostic laboratories a screening method that is relatively quick, straightforward
and cost effective. Using the Chromoprobe Multiprobe system, the
FISH protocol is simplified for a number of reasons. Firstly, the p arm
and q arm telomeres are differentially fluorescently labelled in green
and red; both probe and target DNA are denaturated simultaneously
in a single step; there are no detection or amplification steps; the device
is hybridised overnight; and rapid, formamide-free post hybridisation
stringent washes are used throughout.
Procedure
Sample slide preparation
1. Soak a template slide for 2 min in 100% methanol and polish dry with a
clean soft tissue.
303
opposed to 48 (the p-arms of the acrocentric chromosomes are excluded
because they contain repetitive and ribosomal DNA only and the X and Y
chromosomes share homologous sequences at both pseudoautosomal regions). These probes were produced as cosmid clones with size ranges
from 20-40 kb, making the direct labelling of these for rapid FISH analysis
difficult. In addition, this first generation set contained probes which were
not as close to the end of the chromosome as would be preferred. More
recently, a second generation of probes have been developed. These
probes have been developed as PAC clones and are approximately 100
kb in size making the direct labelling of them possible. These new probes
are of varying distance from the telomere, ranging from 30-450 kb as a
result of the variable length of the TAR.
Standard FISH techniques would require the individual testing of every
telomere probe for each different chromosome, making routine screening
impractical. We have combined the complete set of specific telomere
probes with our Chromoprobe Multiprobe technology to produce a novel
FISH technique to allow the simultaneous hybridisation and analysis of all
probes on one slide. The Multiprobe is a disposable glass device with 24
raised bosses to which each of the 41 telomere probes are reversibly applied following a patented process. Each square thus contains its p-arm
probe in green and its q arm probe in red (except the acrocentric chromosomes which have q- arms only, and the sex chromosomes which share
their telomere specific sequences). This offers cytogenetic diagnostic laboratories a screening method that is relatively quick, straightforward
and cost effective. Using the Chromoprobe Multiprobe system, the
FISH protocol is simplified for a number of reasons. Firstly, the p arm
and q arm telomeres are differentially fluorescently labelled in green
and red; both probe and target DNA are denaturated simultaneously
in a single step; there are no detection or amplification steps; the device
is hybridised overnight; and rapid, formamide-free post hybridisation
stringent washes are used throughout.
Procedure
Sample slide preparation
1. Soak a template slide for 2 min in 100% methanol and polish dry with a
clean soft tissue.