Plastic contaminant masquerades as DNA in mutation detection by denaturing HPLC.

Denaturing high-performance liquid chromatography (DHPLC) has quickly become popular as a tool for the automated discovery of single base substitutions as well as small insertions and deletions (1,2). Under partially denaturing conditions, heteroduplices are retained for a shorter time than their corresponding homoduplices on a DNA separation matrix. We have identified a contaminant emanating from polypropylene PCR tubes that masquerades as DNA in DHPLC assays. While small amounts of plasticizer contaminants are well known anecdotally in DHPLC analyses, the contaminant detected here had a peak area 20–50 times that of the level of PCR products required for analysis. The tube contaminant was identified when using 8 Tube Strips from Axygen Scientific (Union City, CA, USA) while amplifying for 40 cycles a 475-bp fragment of the Paraoxonase gene, PON-1. DHPLC was conducted on a Varian Helix System fitted with a 75-mm Helix Analysis Column packed with C18 alkylated silica (Varian, Walnut Creek, CA, USA). Standard operating procedures were used, utilizing 100 mM triethyammonium acetate (TEAA), pH 7.0, 0.1 mM EDTA as buffer A and 100 mM TEAA, pH 7.0, 0.1 mM EDTA, 25% (v/v) acetonitrile as buffer B (both from Varian), with a gradient of 50%– 68% buffer B over 5.5 min. A similar contaminating peak has been observed using Axygen Scientific 96-Well PCR Plates (personal communication, Me-