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detecting, for example, viral gene expression and furthermore allows the means of

differentiating between latent and active virus (Fig. 6.39). The level of mRNA produc-

tion may also be determined by using a PCR-based method, termed quantitative PCR

(Section 5.10.7).

In many cases the analysis of tissue-specific gene expression is required and again the

PCR has been adapted provide a solution. This technique, termeddifferential display,is

also anRT–PCR-basedsystem requiringthat isolated mRNA be first convertedinto cDNA.

Following this, one of the PCR primers, designed to anneal to a general mRNA element

such as the poly(A) tail in eukaryotic cells, is used in conjunction with a combination of

arbitrary 6–7 bp primers which bind to the 5^0 end of the transcripts. Consequently this

results in the generation of multiple PCR products with reproducible patterns (Fig. 6.40).

Comparative analysisby gelelectrophoresis of PCR products generatedfrom different cell

types therefore allows the identification and isolation of those transcripts that are

differentially expressed. As with many PCR-based techniques the time to identify such

genes is dramatically reduced from the weeks that are required to construct and screen

cDNA libraries to a few days.

Cell with active virus Cell with latent virus

Extract mRNA

Perform RT–PCR

virus-specific primers

Agarose gel electrophoresis

Extract mRNA

Perform RT–PCR

virus-specific primers

Fig. 6.39Representation of the detection of active viruses using RT–PCR.

242 Recombinant DNA and genetic analysis