AluI RFLP Quantitative Assay

A125-bp toxin gene fragment which includes the site of CRM197 mutation was PCR-amplified with oligonucleotide primers：Alu197-F，5'-GACTAAACCTGGTTATGTAGCTTCC-3'（forward primer）；and Alu197-R，5'-CCGCAGCGTCGTATTTATTGTCGGTACTATAAAGC-3'（reverse primer）.

PCR was carried out in a total reaction volume of 100 μL containing 1×PCR buffer，5.5 mmol/L MgCl2，200 μmol/L each dNTP，10 pmol each primer，0.2 ng template genomic DNA，and 5 units Taq DNA polymerase（Boehringer Mannheim）.The PCR mixtures were overlayed with 3 drops of Nujol mineral oil.After predenaturation for 5 min at 95℃，30 PCR cycles were carried out in Perkin-Elmer Cetus DNA Thermal Cycler 480，each cycle including denaturation for 30 s，94℃；annealing 30 s，48℃；and extension 30 s，72℃；followed by a final extension for 3 min at 72℃.

Three microliters of the PCR product was digested with 10 units AluI（New England Biolabs or Boehringer Mannheim）in a total volume of 10 μl overlayed with a drop of Nujol mineral oil to prevent evaporation.Digestions were carried out to completion overnight at 37℃.(https://www.daowen.com)

AluI digests（10 μl）were analyzed by 15%polyacrylamide gel（acrylamide：Bis，19∶1）electrophoresis in 0.5×TBE buffer（45 mmol/L Tris-borate，1 mmol/L EDTA，pH 8.3）using Bio-Rad Mini Protean Ⅱ electrophoresis system.Gels（7 cm long，10 cm wide，1 mm thick）were run at 200 V until the bromophenol blue reached approximately 1 cm from the bottom of the gel.After obtaining the photographs of the ethidium bromide-stained gels with Polaroid positive/negative film No.55，the negatives were scanned using the Personal Densitometer SI（Molecular Dynamics）.

The percentage of CRM197 template in the initial DNA sample was calculated using the formula：