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cell or tissue produces a major protein of the cell a large fraction of the total mRNA

will code for the protein. An example of this are the B cells of the pancreas, which

contain high levels of pro-insulin mRNA. In such cases it is possible to precipitate

polysomes which are actively translating the mRNA, by using antibodies to the

ribosomal proteins; mRNA can then be dissociated from the precipitated ribosomes.

More usually the mRNA required is only a minor component of the total cellular

mRNA. In such cases total mRNA may be fractionated by size using sucrose density

gradient centrifugation. Then each fraction is used to direct the synthesis of proteins

using anin vitrotranslation system (Section 6.7).

6.2.8 Subtractive hybridisation

It is often the case that genes are transcribed in a specific cell type or differentially

activated during a particular stage of cellular growth, often at very low levels. It is

possible to isolate those mRNA transcripts bysubtractive hybridisation. Usually the

the mRNA species common to the different cell types are removed, leaving the cell

type or tissue-specific mRNAs for analysis (Fig. 6.8). This may be undertaken by

isolating the mRNA from the so-calledsubtractor cellsand producing a first-strand

cDNA (Section 6.2.5). The original mRNA from the subtractor cells is then degraded

and the mRNA from the target cells isolated and mixed with the cDNA. All the

complementary mRNA–cDNA molecules common to both cell types will hybridise

leaving the unbound mRNA which may be isolated and further analysed. A more rapid

approach of analysing the differential expression of genes has been developed using

the PCR. This technique, termeddifferential display, is explained in greater detail in

Section 6.8.1.

6.2.9 Cloning PCR products

While PCR has to some extent replaced cloning as a method for the generation of

large quantities of a desired DNA fragment there is, in certain circumstances, still

Subtractor cells Target cells

mRNA

mRNA

unhybridised

cDNA (1st strand)

Analyse unhybridised

mRNA

mRNA

mRNA

cDNA

Fig. 6.8Scheme of analysing specific mRNA molecules by subtractive hybridisation.

204 Recombinant DNA and genetic analysis