1887

Abstract

The locus of harbours two enterotoxin genes ( and ) and three enterotoxin-like genes (, and ). Between the and genes are located two pseudogenes, and , or the or gene. While these two alternative intergenic regions can be distinguished by PCR, to date, DNA sequencing has been the only confirmatory option because of the very high degree of sequence similarity between loci bearing the pseudogenes and the or gene. restriction enzyme digestion of genomic regions encompassing the locus from the 3′ end of the gene through the 5′ first quarter of the gene allowed pseudogene- and - or -bearing loci to be distinguished by PCR-RFLP. Experimental application of these findings demonstrated that endonuclease dIII cleaved PCR amplimers bearing pseudogenes but not those with a or gene, while - or -bearing amplimers were susceptible to cleavage by endonuclease I, but not by endonuclease dIII. The restriction enzyme I cleaved - or -harbouring amplimers at a unique restriction site created by their signature 15 bp insertion compared with pseudogene-bearing amplimers, thereby allowing distinction of these loci. PCR-RFLP analysis using these restriction enzymes provides a rapid, easy to interpret alternative to DNA sequencing for verification of PCR findings on the nature of an locus type, and can also be used for the primary identification of the intergenic locus type.

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2007-02-01
2019-10-21
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