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proteins may be detected by polyacrylamide gel electrophoresis or by immunological

detection usingwestern blotting. Recently oligonucleotide PCR primers have been

designed to incorporate a promoter for RNA polymerase and a ribosome-binding site.

When the so-calledexpression PCR(E-PCR) is carried out the amplified products are

denatured and transcribed by RNA polymerase after which they are translatedin vitro.

The advantage of this system is that large amounts of specific RNA are synthesised thus

increasing the yield of specific proteins (Fig. 6.35).

6.7.1 Production of fusion proteins

For a foreign gene to be expressed in a bacterial cell, it must have particular promoter

sequences upstream of the coding region, to which the RNA polymerase will bind

prior to transcription of the gene. The choice of promoter is vital for correct and

efficient transcription since the sequence and position of promoters are specific to a

particular host such asE. coli(Section 5.5.4). It must also contain a ribosome-binding

site, placed just before the coding region. Unless a cloned gene contains both of these

sequences, it will not be expressed in a bacterial host cell. If the gene has been

produced via cDNA from a eukaryotic cell, then it will certainly not have any such

sequences. Consequently, expression vectors have been developed which contain

promoter and ribosome-binding sites positioned just before one or more restriction

Primer 1 Primer 2

T7

promoter

Start

codon

RestrictionRBS UTR

cloning site

Stop codon

5 

3  5 

3 

Amplify gene by PCR with primers 1 and 2

PCR product with potential for protein production

Transcription and translation

Restriction cloning site

Fig. 6.35Expression PCR (E-PCR). This technique amplifies a target sequence with one promoter that

contains a transcriptional promoter, ribosome binding site (RBS), untranslated leader region (UTR) and start

codon. The other primer contains a stop codon. The amplified PCR products may be used in transcription

and translation to produce a protein.

236 Recombinant DNA and genetic analysis