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Plastic test tube chemicals skew DNA data

Sep 2010
Margaret W. Bushee,

Nucleic acids such as DNA and RNA absorb UV wavelengths, so genetic research is dependent upon accurate data gathered in this spectral range. Typically, absorbance spectroscopy measurements at 220- and 260-nm wavelengths are used to determine concentrations of proteins and DNA, respectively.

Therefore, during a recent study on clay nanoparticles as a potential delivery system for DNA, graduate student Michael H. Robson of Texas State University took notice when some absorbance spectroscopy data he had compiled appeared inconsistent. He observed that, after the aqueous solution had been centrifuged at a high speed – and consequently heated – for 30 minutes or more, the readings at the 260-nm wavelength were as much as three times higher than before the centrifugation.

He suspected that the chemicals in the polypropylene sample containers, called microfuge tubes or microtubes, were the culprit. Modeled after traditional glass test tubes and used worldwide for all types of laboratory research, microtubes contain a variety of chemical additives that function as antioxidants. Robson surmised that the heated solution was causing the chemicals to leach out and absorb UV wavelengths, skewing what was ostensibly the DNA concentration data.

A recent study at Texas State University in San Marcos determined that polymer microtubes leach chemicals into heated solutions, where they absorb wavelengths that are used to identify genetic material. Previously recorded DNA data may have to be re-evaluated.

The study, led by L. Kevin Lewis and co-written by Gary W. Beall, both associate chemistry professors at the university, was published in the Jan. 12, 2009, issue of Biomacromolecules.

To follow up on Robson’s hunch, the team designed another study using 10 varieties of microtubes from nine manufacturers. The 1.5-mL vessels were filled with 1 mL of deionized water and heated for 30 minutes in a 100 °C heating block. Absorbance spectroscopy was used to take 220- and 260-nm readings from each sample.

The researchers established that the microtubes could be divided into two categories according to their absorption spectra. Most microtubes had a dominant peak at approximately 220 nm, whereas the smaller set had a dominant peak at approximately 260 nm. They also found that, in all of the microtubes, leaching escalated when the tubes were heated to higher than 37 °C and when organic solvents were used. Both are common procedures during genetic research. The team determined that the data at 220- and 260-nm wavelengths was both time- and temperature-dependent.

The latter study was published in the April 2010 issue of BioTechniques.

Even though the research left many questions unanswered, the investigators felt compelled to share their findings because the data from many genetic studies may have to be reconsidered. “We felt that it was important to go ahead and publish, since the effect could impact a lot of researchers’ work,” Beall said. “There indeed could be a substantial amount of data in the literature that needs to be examined for data bias.” As for forensic DNA testing, however, he noted that “it should not be affected since it is a different type of test.”

Beall commented that the researchers’ own methods have “most definitely” been influenced by their recent study. “Our work now uses glass when possible, but certainly will use the tubes that we found to leach the least. We will always run these with a background correction to account for the leaching.”

The process of removing some of the constituents of a glass surface by chemical action.
A material whose molecular structure consists of long chains made up by the repetition of many (usually thousands) of similar groups of atoms.
absorbance spectroscopyAmericasBasic SciencebiochemistryBiomacromoleculesBiophotonicsBioScanBioTechniqueschemicalsclay nanoparticlesDNAGary W. BeallL. Kevin LewisleachingMargaret W. BusheeMichael H. RobsonmicrocentrifugemicrofugenanoNewsnucleic acidpolymerpolypropyleneRNASan MarcosTest & Measurementtest tubeTexas State Universityultravioletlasers

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