Evelyn McGown, Ph.D. and Michael Su, M.S.
Molecular Devices Corporation, 8/00
DNA measurements are important for many aspects of molecular biology. Of thespectrophotometric methods available, UV absorbance measurement at 260 nm isthe simplest and has recently gained widespread popularity in the microplateformat.1 However, A260 measurements do not distinguish between dsDNA,ssDNA and RNA. Also, they are relatively insensitive (limit of quantitationapproximately 250 ng/mL in traditional spectrophotometers2,3 and approximately 100 ng/well in a 96-well microplate1). Below these levels, analternative method must be used such as the PicoGreen reagent from Molecular Probes, Inc.4 The latter reagent binds to dsDNA with high affinity and becomesfluorescent. The limit of detection in a microplate well is approximately 50 pgdsDNA per well.4 Recently, a luminometric method intended for use in standardtube luminometers was introduced by Promega Corporation.5 The DNAQuant is specific for linear dsDNA (including PCR fragments) and is not affected bycontaminants (e.g. ssDNA , RNA, protein) which interfere with other methods.This method has been adapted to Molecular Devices Lmax microplateluminometer with a resultant lower limit of approximately 5 pg DNA per well.The method and results are described below.
PRINCIPLE OF ASSAY
The DNAQuant uses a coupled enzyme system to produce ATP in an amount proportional to the amount of dsDNA present, followed by addition of luciferin/luciferase reagent to generate light in proportion to the amount of ATP (Figure 1).The coupled set of reactions consists of a pyrophosphorylation andtransphosphorylation (pyro/transphosphorylation). The pyrophosphorylationreaction uses T4 DNA polymerase in the reverse direction. In the presence ofpyrophosphate and dsDNA, T4 DNA polymerase catalyzes the release ofdeoxynucleotide triphosphates (dNTPs) from the 3 termini of the DNA strands.
The transphosphorylation reaction is catalyzed by the enzyme nucleoside diphosphate kinase (NDPK). In this reaction, the terminal phosphate of the dNTP is transferred to ADP to form ATP. The ATP is then measured using a luciferin/ luciferase reagent to produce light in proportion to the amount of ATP which, in turn, is proportional to the amount of dsDNA.
1. Lmax microplate luminometer (Molecular Devices Corp.)
2. DNAQuant DNA Quantitation System, Product No. K4000; Promega Corporation. 608-277-2516.
3. Solid white microplates; e.g. CorningCostar Cat. No. 3912, Tel: 1800492 1110)
PREPARATION OF REAGENTS AND STANDARDS
The instructions supplied with the kit (Promega Technical Bulletin No. 278) werefollowed, except for minor modifications to accommodate a 96-well format.
1. The enzyme and buffer solutions were thawed and kept on ice.
2. TE buffer (10 mM Tris and 1 mM EDTA in sterile, deionized water, pH adjusted to 7.4) was sterilized by filtration through an 0.2 m filter.
3. The luciferin/luciferase (ENLITEN L/L Reagent) was reconstituted by adding 12 mL of L/L Reconstitution Buffer to the vial, gently inverting several times and allowing to stand at room temperature for at least 30 min.
4. The Master Mix was prepared immediately before use by adding the following components in the order stated: 465 L DNA quantitation buffer solution, 15 L sodium pyrophosphate, 3 L NDPK enzyme solution, 27 L sterile, deionized water, 30 L T4 DNA polymerase.
5. The DNA standards were prepared by first diluting 50 L DNA stock standard (5 ng/mL; supplied in the kit) with 450 L TE buffer, followed by serial 1-to-2 dilutions i n TE buffer. The diluted standards (3.9 to 250 pg/L) were kept on ice until use.
The following procedure differs from the original, in that the DNA and master mix were incubated in microplate wells instead of microcentrifuge tubes. Then instead of transferring aliquots to separate tubes, the ENLITEN L/L Reagent was added directly to each microplate well by automatic instrument injection immediately before reading.
6. To each well of the microplate were transferred 2 L diluted DNA standard (or buffer blank) and 18 L Master Mix. The final amounts of DNA were 7.8 to 500 pg/well. The plate was covered and incubated 10 minutes in the dark at ambient temperature.
7. The Lmax was set up to inject 100 L/well, followed by a 3-second delay and a 1-second read. The P-injector was primed with ENLITEN L/L Reagent.
8. After the 10 minute pre-incubation, the plate was placed into the drawer of the Lmax and the injection/read cycle initiated.
EXAMPLE OF DNA RESULTS OBTAINED ON THE LMAX
The DNAQuant package insert claims a linear range of 20 to 1000 pg/reaction mixture, and this was confirmed by the results shown in Figure 2. The lower end of the curve is shown in Figure 3. The calculated limit of detection (amount producing a signal higher than 3 positive SD of the blank values above zero) was approximately 5 pg/well. Thus the sensitivy of the assay in the Lmax is as good as, if not better than, that reported for a tube-type luminometer.
With a lower limit of 5 pg DNA/well, the DNAQuant clearly offers a sensitive method for measuring dsDNA. It offers the advantage that ssDNA, RNA and protein do not interfere. However, one drawback is the fact that the recommended sample volume is small compare d to the volumes of the reagents. With a sample volume of 2 L, the lower limit corresponds to a DNA concentration of 2.5 ng/mL in the original sample. Lower concentrations probably cannot be measured by this method without pre-concentration or reformulation of the reagents.
The Lmax microplate luminometer gives a lower limit of 5 pg DNA/well with the DNAQuant DNA quantitation system. These results are as good as, if not better than, results obtained on a standard tube luminometer. The Lmax offers the advantage that the addition of luciferin/luciferase reagent and subsequent read can be automated so that variability due to differences in timing of the flash luminescence reaction can be eliminated.
1. McGown, E.L. 2000. UV Absorbance Measurements of DNA in Microplates. BioTechniques. 28, 60-64.
2. Gallagher, S.R. 1994. Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy. Current Protocols In Molecular Biology, Vol. 3. John Wiley & Sons, pp. A3.D.1A3.D3.
3. Sambrook, J., E. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. p. E.5.
4. Molecular Probes, Inc.: www.probes.com.
5. Promega Corporation: www.promega.com