Engineering Deletions and Insertions by PCR

PCR is widely used to generate DNA cassettes that can be introduced into chromosomes by homologous recombination. In this procedure, a convenient marker gene, usually an antibiotic resistance gene, is inserted into the chromosome of the host organism where it replaces any chosen gene. In order to target the incoming cassette to the correct location it must first be flanked with DNA sequences homologous to DNA both upstream and downstream of the chosen gene. This is done by using PCR primers that overlap the resistance cassette and also contain about 40-50 bp of DNA homologous in sequence to the target location (Fig. 23.19). The cassette is transformed into the host organism and is inserted into the chromosome by homologous recombination. Antibiotic resistance is then used to select those organisms that have gained the cassette. This approach may be used to generate deletions of any desired chromosomal gene and works especially well in yeast and bacteria.

directed mutagenesis Deliberate alteration of the DNA sequence of a gene by any of a variety of artificial techniques

Resistance gene

Downstream primer

Downstream primer

Assemble by overlap pcr

Resistance cassette with homologous ends

Resistance cassette with homologous ends

Transform into target cell

FIGURE 23.19 Generation of Insertion or Deletion by PCR

In the first step, a specifically targeted cassette is constructed by PCR. This contains both a suitable marker gene and upstream and downstream sequences homologous to the chromosomal gene to be replaced. The engineered cassette is transformed into the host cell and homologous crossing over occurs. Recombinants are selected by the antibiotic resistance carried on the cassette.

A collection of yeast strains deleted for all approximately 6,000 known genes has been generated by this procedure. Each strain has had a single coding sequence replaced by a cassette comprising the npt gene plus a barcode sequence. The npt gene encodes neomycin phosphotransferase which confers resistance to neomycin and kanamycin on bacteria and resistance to the related antibiotic geneticin on eukaryotic cells, such as yeast. A barcode sequence is a unique sequence of around 20 bp that is included as a molecular identity tag. Each insertion has a unique barcode sequence allowing it to be tracked and identified. Such barcode or zipcode sequences are increasingly being used in high volume DNA screening projects where it is necessary to keep track of many similar constructs.

Clearly, the above procedure also generates an insertion of whatever gene is carried on the cassette. Thus any foreign gene may be inserted by this approach. There is no need to delete a resident gene if the objective is the insertion of an extra gene. All that is necessary is that the incoming gene must be flanked with appropriate lengths of DNA homologous to some location on the host chromosome.

Upstream primer

Upstream primer

Resistance gene

V' Homologous crossing over\'

Gene to be deleted

Chromosome with deletion

Resistance gene

Barcode sequence

Traces of DNA can be amplified by PCR and used for diagnosis or forensic analysis.

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