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Microplate-based Spectrophotometric Quantification of dsDNA Using a Variety of Methods下載
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June 27, 2018
DNA is used for a variety of downstream applications ranging from PCR to sequencing. Standard methods of DNA quantification post-isolation include spectrophotometric absorption at 260 nm and fluorescence when DNA is chelated with a binding dye. The most common method remains spectrophotometry due to cost and ease of use. Traditionally measurements have been done in a cuvette-based spectrophotometer with a 1-cm pathlength vessel. Yet for analysis of a large number of nucleic acid samples, a microplate format is preferred.
All double-stranded DNA (dsDNA) standards were created by preparing a 1:2 serial dilution series of a concentrated stock in TE buffer (tris-EDTA, pH=7.0). Measurements were performed in triplicate in a 100 μL volume.
Herring Sperm DNA, cat # D6898 was purchased from Sigma-Aldrich. Quant-iT™ PicoGreen Reagent, cat # P11495 was purchased from Thermo-Fisher. Solid 96-well black microplates, (cat # 3915), were from Corning. Herring sperm DNA was diluted to 2 μg/mL with TE (10 mM Tris, 1 mM EDTA, pH 7.5) as the diluent. The final concentration was confirmed using 260 nm absorbance. A series of dilutions ranging from 0.0 to 2000 ng/mL of purified Herring Sperm was made using TE and 100 μL aliquots pipetted into microplate wells. Equal amounts (100 μL) of working PicoGreen quantitation reagent were mixed and incubated for 10 minutes at room temperature, protected from light. Working PicoGreen reagent was prepared by diluting the concentrated DMSO-PicoGreen stock solution 1:200 with TE according to the manufacturers’ recommendations.
The Beer-Lambert Law relates the absorption and concentration via the following equation where A = log Io/I = εlc. ε refers to the extinction coefficient of the analyte, l is the pathlength (cm) and c is the concentration of the analyte (ng/μL).
BioTek’s reader control and data analysis software, Gen5™, has built-in methods for path length correction to 1 cm for samples in microplates where path length is defined by the volume of the sample added. The calculation is performed by measuring the absorbance peak of water at 977 nm and 900 nm, the difference is then divided by 0.18, which is the absorbance of water at 1 cm, equaling the sample’s path length. The Synergy™ LX Multi-Mode Reader with absorbance monochromators was used to measure absorbance at 230, 260, 320, 900 and 977 nm in a single protocol to allow automated path length correction, DNA quantification and ratiometric sample purity assessment.
Fluorescence was determined using a Synergy LX Multi-Mode Reader. The onboard software and touchscreen interface were used to select the appropriate filter cube, define shaking parameters, add delays, and select well locations and protocol parameters. A GFP filter cube (EX 485/20 nm, EM 528/25 nm and 510 nm cutoff dichroic mirror) was used for all measurements. Auto gain was selected with Extended Dynamic Range. A 30-second orbital shake was performed followed by a 10 min. delay for incubation at room temperature. Alternatively, the instrument can be controlled and analysis performed in the Gen5 software or data imported for analysis.
Results and Discussion
The use of absorbance and fluorescence methods for quantification of double-stranded DNA allows a wide range of concentrations to be analyzed. UV absorbance is the most common and easiest method requiring no additional reagents but can suffer from limits of detection especially when working with sub-microgram per milliliter DNA samples. Here we show good linearity to the low μg/mL concentrations of DNA (Figure 1). The data indicate a limit of detection of ~8 μg/mL using UV absorbance or ~ 0.8 μg in the 100 μL volume. The ability to take multiple UV absorbance readings at different wavelengths provides a means to verify sample purity as well. Typically the ratio of A260/A280 is used with values between 1.8 – 2.0 indicative of a pure sample. It is typical that for very low concentration samples the ratio is typically lower than the expected value due to a greater influence of the A280 measurement in the calculation (Table 1).
Figure 1. Absorbance measurements. A dilution series of herring sperm DNA was prepared ranging from 0 – 550 μg/mL in TE buffer. Standards were analyzed in triplicate at a volume of 100 μL in a standard 96-well microplate format. A260 measurements are pathlength corrected.
Table 1. A260/A280 ratio. The ratio of absorbance measurements at 260 and 280 nm can be used to access the purity of a sample. Typical ratios for pure DNA range from ~1.8 – 2.0.
The use of fluorescence methods can extend the range of quantification to picogram quantities of DNA in a standard microplate assay format. Here we show the ability to measure low ng/mL quantities of DNA (Figure 2). The data indicate a limit of detection of ~121 pg/mL using fluorescence or ~ 12.1 pg in the 100 μL volume.
Figure 2. Fluorescence assay. A dilution series of herring sperm DNA was prepared ranging from 0 – 1000 ng/mL in TE buffer. Standards were analyzed in triplicate in a standard 96-well microplate format.
The Synergy™ LX Multi-Mode Reader provides the most common detection technologies used in biological research including absorbance, fluorescence and luminescence detection. Here we demonstrated its utility to quantify DNA in microplates using intrinsic UV detection and a fluorescence assay to extend the dynamic range of measurable concentrations.