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6.8.2 Analysing genesin situ

Gross chromosomal changes are often detectable by microscopic examination of the

chromosomes within a karyotype (Section 5.3). Single or restricted numbers of base

substitutions, deletions, rearrangements or insertions are far less easily detectable but

may induce similarly profound effects on normal cellular biochemistry.In situhybrid-

isation makes it possible to determine the chromosomal location of a particular gene

fragment or gene mutation. This is carried out by preparing a radiolabelled DNA or RNA

probe and applying this to a tissue or chromosomal preparation fixed to a microscope

slide. Any probe that does not hybridise to complementary sequences is washed off and

an image of the distribution or location of the bound probe is viewed by autoradiog-

raphy (Fig. 6.41). Using tissue or cells fixed to slides it is also possible to carry outin situ

PCR and qPCR. This is a highly sensitive technique where PCR is carried out directly on

the tissue slide with the standard PCR reagents. Specially adapted thermal cycling

machines are required to hold the slide preparations and allow the PCR to proceed.

Total cellular mRNA

cDNA synthesis

PCR amplification

(Arbitrary primers 6 to 7 bp

used in various combinations)

Arbitrary primer

AAAAA

TTTT

Anchored primer

Multiple PCR products

separated by gel electrophoresis

Autoradiograph

Comparative analysis of

differentially expressed genes

Fig. 6.40Analysis of gene expression using differential display PCR.

243 6.8 Analysing genes and gene expression