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Interference with spectrophotometric analysis of nucleic acids and proteins by leaching of chemicals from plastic tubes
 
L. Kevin Lewis, Michael H. Robson, Yelena Vecherkina, Chang Ji, and Gary W. Beall
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GC–mass spectrometry

An Agilent Technologies (Santa Clara, CA, USA) model 6890N gas chromatograph (GC) equipped with a model 5973N mass selective detector was utilized for analysis of the leachate for compounds with molecular masses up to 200. Analytes were separated using an Agilent Technologies HP-5MS 30 mm × 0.25 mm capillary column with a 5% crosslinked phenylmethylsiloxane stationary phase. The oven temperature was held initially at 45°C for 6 min and then ramped to 200°C at 8°C/min. The split ratio was 20:1.

Electrospray ionization mass spectrometry

Positive-ion and negative-ion electrospray ionization experiments were performed using the conventional electrospray ionization source of an LCQ Classic (Thermo-Fisher, Waltham, MA, USA) operating at m/z range of 200–2000. Sample solutions were introduced into the electrospray ionization source using a 5-µL sample loop. Methanol with a flow rate of 50 µL/min was used as the push solvent. The mass spectrometer experimental conditions were as follows: sheath gas flow rate, 75 (arbitrary units); aux gas flow rate, 20; and capillary temperature, 200°C. Spray voltages were 5 kV (positive ion mode) and 3.5 kV (negative ion mode).

Results and discussion

As part of a recent spectrophotometric study of the efficiency of sedimentation of DNA and clay particles by centrifugation (14), we observed that prolonged high-speed centrifugation and concomitant warming of plastic microtubes caused an unexpected increase in UV absorbance at 260 nm. To investigate the phenomenon more comprehensively, absorbances of several commercially available brands of standard microtubes containing 1 mL deionized water were analyzed before and after heating the tubes for 30 min in a 100°C heating block. New absorbance peaks appeared at approximately 220 and 260 nm upon heating, producing two consistent patterns (Figure 1A). Most tubes displayed the type 1 spectrum, with a large peak near 220 nm (218 nm maximum) and smaller peak near 260 nm (259 nm maximum). In a minority of tubes, the peak near 260 nm was dominant. No other absorbance peaks were observed within the wavelength range of 200–600 nm in any of the tubes tested.

A representative tube from the most common group—type 1—was used for subsequent studies of leaching from standard 1.5-mL tubes except where noted otherwise. The magnitude of leachate peak absorbance could be substantial (up to 1.0–2.0 absorbance units) and was temperature-dependent (Figure 1B, top panel). Modest chromophore leaching was detectable after 30 min at 37°C, the temperature used for most enzyme-catalyzed reactions in biochemistry, but seepage was strongest at higher temperatures (70°C and 95°C). These high temperatures are often employed to heat-inactivate enzymes, denature DNA, and perform other reactions such as PCR that involve thermostable enzymes (described below). The effect was time-dependent and rapid, being readily observable <5 min after exposure to elevated temperatures (Figure 1B, bottom panel).

Nucleic acids absorb light strongly at 260 nm and absorbance at this wavelength is commonly employed to detect and quantitate DNA (1). Proteins absorb UV light at 220 nm due to the presence of double bonds within amino acid carbonyl groups. Most proteins also absorb light at 280 nm, with peak height at 280 nm dependent primarily upon the fraction of tryptophan and tyrosine amino acids within the protein. To examine how biomolecule absorbance spectra are shifted by heating in microtubes, aqueous solutions containing a single-stranded DNA oligonucleotide (25 nucleotides in length) were scanned before and after heating for 30 min. As shown in Figure 1C, heating caused a substantial increase in absorbance at 260 nm in type 1 tubes. This increase, from 0.31 to 0.48, is equivalent to an apparent change in DNA concentration from 10.2 to 15.8 µg/mL, an overestimate of 55%. In the type 2 tubes analyzed in Figure 1C, a larger increase in absorbance was observed (0.24 to 0.96), suggesting a change from 7.9 to 31.7 µg/mL, resulting in an overestimate of 300%. No increase in absorbance occurred when heating was performed using a glass test tube as control.

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