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Heteroduplex Detection with a Plant DNA Endonuclease for Standard Gel Electrophoresis
Manuela F. Scaffino, Andrea Pilotto, Stavros Papadimitriu, Marco Sbalzarini, Silvia Ansaldi, Marta Diegoli, Emanuele Porcu, Maurizia Grasso, Agnese Brga and Eloisa Arbustini

CEL I is a celery-derived endonuclease that cuts both strands of the heteroduplex DNA on the 3’ side of any mismatch site. It has been proven to recognize heteroduplexes, both insertions/deletions and single base substitutions, in plant, human and animal genes at a rate of efficiency that varies with the sequence of the mismatch. We tested the sensitivity of CEL I (Transgenomic-Omaha, NE), marketed under the name SURVEYORTM Nuclease, in 19 amplicons carrying known mutations previously identified by sequencing in 13 exons of 8 different genes (FBN1, KCNQ1, Cx26, TAZ G4.5, SCN5A, TNNT2, MYBPC3, HFE). Thirteen wild-type amplicons were used as negative controls. We also tested CEL I specificity for the type of gene defects (deletions, insertions and single base substitution: purine vs. pyrimidine and vice versa). Amplicons ranging 154-400 bp in size were obtained by proofreading-Taq based PCR. The PCR conditions were mantained as in routine. Heteroduplexes were generated by a standard thermal cycler program: 95°C for 10 min; 95°C to 85°C(-2°/sec); 85°C to 25°C(-0.1°/sec); 4°C. CEL I endonuclease digestion was performed with 200 ng of heteroduplex DNA and controlled according to the manufacturer’s SURVEYOR Nuclease Kit protocol. Electrophoresis was run on 2% Nusieve/1% Agarose gel. CEL I detected 17 of the 19 heteroduplexes mutations (sensitivity 90%): 3/3 deletions, 2/2 insertions and 12/14 single base substitutions. Results were confirmed in 3 sets of repeated experiments. The wild-type samples did not generate any false positive results. CEL I is a simple, fast, sensitive and reproducible tool for heteroduplexes detection.

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