Tuesday, September 29, 2009

DNA Purity Verification in Microplates Using the A260/A280 Ratio

A common practice in molecular biology is to perform a quick assessment of the purity of nucleic acid samples. While purification of nucleic acid can be accomplished by a number different techniques such as protease digestion followed by organic extraction or by column chromatography, assessment of the purity is almost always carried out by ratiometric spectroscopy at 260 nm and 280 nm. Although this procedure was first described by Warburg and Christian [1] as a means to measure protein purity in the presence of nucleic acid contamination, it is most commonly used today to assess purity of nucleic acid samples. Traditionally this procedure has been performed in spectrophotometers one or two samples at a time using quartz cuvettes. While this method is effective the number of samples that can be determined using this method is limited. The advent of UV/Vis microplate readers and the availability of transparent microplates has allowed for numerous samples to be tested simultaneously.

It is important to note that the A260/A280 ratio is only an indication of purity [2, 3] rather than a precise answer. Pure DNA and RNA preparations have expected A260/A280 ratios of ³1.8 and ³ 2.0 respectively [3] and are based on the extinction coefficients of nucleic acids at 260 nm and 280 nm. Although the A260/A280 ratio is relatively insensitive to change and seemingly useless when DNA/protein mixtures are experimentally tested, the utility of this procedure becomes apparent when nucleic acids are purified from tissue or blood. Tissue samples and to a lesser extent whole cells have a protein content that greatly exceeds that of nucleic acid on a weight basis and purification of samples to a A260/A280 ratio represents an enrichment of nucleic acid that could be as much as 1 million fold.

There are several factors that may affect A260/A280 ratios. The 260 nm measurements are made very near the peak of the absorbance spectrum for nucleic acids, while the 280 nm measurement is located in a portion of the spectrum that has a very steep slope. As a result, very small differences in the wavelength in and around 280 nm will effect greater changes in the A260/A280 ratio than small differences at 260 nm. Consequently, different instruments will result in slightly different A260/A280 ratios on the same solution due to the variability of wavelength accuracy between instruments. Individual instruments, however, should give consistent results. Concentration can also affect the results, as dilute samples will have very little difference between the absorbance at 260 nm and that at 280 nm. With very small differences, the detection limit and resolution of the instrument measurements begin to become much more significant. Pure DNA has an expected A260/A280 ratio of 1.85-2.0. As such the 280 nm value is approximately half that of the 260 nm value. With decreasing nucleic acid concentrations one can imagine that the 280 nm value will eventually be below the detection limit of the microplate reader before the 260 nm value. This is exacerbated in microplates, where the pathlength of the absorbing material is usually less than the 1 cm found with standard cuvettes. Subtraction of background absorbance caused by the microplate of the sample buffer is critical with microplate measurements. Besides the actual measurement the most critical element in the determination of the A260/A280 ratio is the subtraction of the background absorbance of the microplate. This is often referred to as blanking. Blanking requires that one or two wells be filled with buffer only and their respect absorbance values at 260 nm and 280 nm be subtracted from all of the experimental samples. Failure to subtract the background will result in false but believable results at high nucleic acid concentrations and totally aberrant results at low nucleic acid concentrations.

The use of UV/transparent microplate allows for a major increase in throughput for this routine assay. Instead of laboriously reading samples in a cuvette, cleaning the cuvette and loading the next sample, one can read 96 or 384 samples (minus one or two wells for blanking) in minutes instead of hours. Making A260/A280 determinations in microplates will not be any more accurate than cuvettes, but it will save you hours of time.

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References

(1) Warburg, O. and W. Christian (1942) Isolation and crystallization of enolase. Biochem. Z. 310:384-421.
(2) Glasel, J.A. (1995) Validity of Nucleic Acid Purities Monitored by A260/A280 Absorbance Ratios, Biotechniques 18:62-63.
(3) Maniatis T., E.F. Fritsch, and J. Sambrook (1982) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, NY.



By: BioTek Instruments



1 comment:

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