Quantitative restriction fragment length polymorphism: a procedure for quantitation of diphtheria toxin gene CRM197...

Here we present an assay for quantitation of a particular gene allele in DNA mixtures by means of restriction fragment length polymorphism (RFLP) in combination with polymerase chain reaction (PCR). We applied the quantitative RFLP principle for estimation of the relative amount of diphtheria toxin gene CRM197 allele in Corynebacterium diphtheriae culture DNA samples. The procedure is based on PCR-mediated generation of an artificial AluI restriction site specifically with the CRM197 DNA template. After AluI digestion of the PCR product and polyacrylamide gel electrophoresis of the restriction fragments, the percentage of CRM197 template in the initial DNA sample was determined by scanning a gel negative. The method was shown to give a linear response when applied to template mixtures containing different amounts of CRM197 reference template. For samples where non-CRM197 DNA was detected by AluI RFLP, we designed a further allele-specific PCR assay to determine whether the non-CRM197 template portion was the wild-type toxin gene allele.