CRM197 Quantitation in Template Mixtures by AluI R...
To determine whether the Alul RFLP approach would accurately estimate the relative amount of CRM197 toxin gene mutant allele in DNA samples，template mixtures containing different percentages of wild-type and CRM197 mutant reference DNAs were subjected to Alul RFLP analysis.
Typical gel patterns in AluI RFLP assay as shown in Figure 2.The scan data，summarized in Table 1，indicate high reproducibility of the AluI RFLP assay in four independent experiments.The percentage of（71+34 bp）AluI fragments is directly proportional to the percentage of CRM197 in the template mixture，as demonstrated by the linear regression curve in Figure 2（b）（R2=0.997）.The detection limit of the assay is 5%of the particular template DNA in the sample（Figure 2（a））.With lower template amounts，the allele-specific restriction fragments were barely detectable on the gel.
Figure 2 CRM197 quantitation in CRM197+wild-type template mixtures by AluI RFLP
（a）AluI restriction fragments separated in 15%polyacrylamide gel.Lane 1：nondigested 125 bp PCR product.Lane 2—Lane 14：AluI fragments of the PCR products generated with 100%，99%，95%，90%，80%，70%，60%，50%，40%，30%，20%，10%，and 0%of CRM197-Ref.in the template mixture.Lane 15：Molecular weight marker V（Boehringer Mannheim）（b）Linear regression curve representing correlation between percentage CRM197 in the template mixture and the percentage of（71+34 bp）AluI fragments(https://www.daowen.com)
