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This allows the localisation and identification of, for example, single copies of intracel-

lular viruses and in the case of qPCR the determination of initial concentrations of

nucleic acid.

An alternative labelling strategy used in karyotyping and gene localisation isfluores-

centin situhybridisation(FISH). This method sometimes termed chromosome painting

is based onin situhybridisation but in which different gene probes are labelled with

different fluorochromes, each specific for a particular chromosome. The advantage of this

method is that separate gene regions may be identified and comparisons made within the

same chromosome preparation. The technique is also likely to be highly useful in genome

mapping for ordering DNA probes along a chromosomal segment (Section 6.9).

6.8.3 Analysing promoter–protein interactions

To determine potential transcriptional regulatory sequences genomic DNA fragments

may be cloned into specially devised promoter probe vectors. These contain sites for

insertion of foreign DNA which lies upstream of a reporter gene. A number of reporter

genes are currently used, including thelacZgene encodingb-galactosidase, theCAT

geneencoding chloramphenicol acetyl transferase (CAT) and theluxgene which

produces luciferase and is determined in a bioluminescent assay. Fragments of DNA

potentially containing a promoter region are cloned into the vector and the constructs

Cell Fixation Method

Pretreatment of cell

Post-Fixation Treatment

Cell permeabilisation

Addition of Labelled Probe

Hybridisation conditions required

Wash Excess Probe from Section

Detection of Hybridised Probe

Radioactive/Non-radioactive detection

Fig. 6.41General scheme forin situhybridisation.

244 Recombinant DNA and genetic analysis