Principle of Alul RFLP Assay

In the AluI RFLP assay，a 125-bp tox gene fragment including the site of CRM197 point mutation was PCR-amplified with oligonucleotide primers Alu197-F and Alu197-R.These primers were designed to generate artificial AGCT Alul restriction sites in the PCR product.Introduction of an artificial restriction site by site-directed mutagenesis for RFLP assay purposes is often applied when the normal or mutant sequence，such as CRM197，does not form any known endonuclease restriction site［14］.Amplification with Alu197-F will generate a control AluI site at the 5'-end of the PCR product.Amplification with Alu197-R will generate the AluI site only with the CRM197 template，but not with wild-type or with a template other than CRM197（see Figure 1）.After AluI digestion of the PCR product，the expected RFLP patterns in polyacrylamide gel electrophoresis are 105+20 bp（control fragment）for the non-CRM197 DNA template，and 71+34+20 bp（control fragment）for the CRM197 DNA template（see Figure 1）.

Figure 1　Principle of AluI RFLP approach：restriction map of 125-bp PCR product and sequences generated with different templates(https://www.daowen.com)