Quantitative RFLP: A Procedure for Quantitation of Diphtheria Toxin Gene CRM197 Allele

Diphtheria toxin protein secreted by pathogenic strains of Corynebacterium diphtheriae is the causative agent for diphtheria in humans. CRM197 protein that is used for vaccination against diphtheria is a non-toxic mutant derivative of the diphtheria toxin resulting from G-->A transition in the tox gene encoding a GLY-->GLU substitution in position 52 of the protein amino acid sequence. Considering the possibility of CRM197 allele reversion to wild-type tox gene sequence during cultivation of bacteria, it is extremely important to monitor the genetic stability of CRM197 mutation in process of protein production. For that purpose we have designed a method 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 have applied the quantitative RFLP principle for estimation of the relative amount of diphtheria toxin gene CRM197 allele in DNA samples isolated from the cultures of Corynebacterium diphtheriae. The procedure presented in Figure1, is based on PCR-mediated generation of an artificial Alu I restriction site specifically with the CRM197 DNA template. After Alu I 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 as shown in Figure 2 and calculated as shown in Figure 3. The method produced a linear response in the range of 0-100% of CRM197 reference template DNA in the sample (Figure 4) with a detection limit of 5% (Figure 3).

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