001DNA measurements in low volume samples, microplates and cuvettes on the SPECTROstar Nano

Introduction

Absorbance measurements
Definition absorbance
Generally, most substances in solution are able to absorb light at a special wavelength or wavelength range. This means that after sending a defined amount of light (I₀) through the solution, a reduced amount of light (I) will be detected afterward.

There is a direct linear relation between the absorbance and the concentration of the solute, up to certain limits. This relation is shown in the Beer-Lambert or Beer’s law.

b = pathlength [cm]
c = concentration of absorbing substance in solution [mol/l or M]
e = substance-specific constant [cm-1 M-1] (extinction coefficient)

In this technical application note, the direct determination of DNA using absorbance is explained.

DNA
Present in all living organisms, DNA nucleotides consist of a sugar backbone, a base (thymine, guanine, adenine, cytosine), and a phosphate group. The nitrogen rich bases absorb light at 260 nm, this wavelength can be used to determine the DNA concentration. As DNA is so highly studied, the extinction coefficients for dsDNA, ssDNA, and for RNA are widely known. The reciprocal value of the coefficient at a 1 cm pathlength can be used as a  factor to determine the concentration of nucleic acids.

Table 1: Reciprocal value of extinction coefficients for nucleic acids.

¹ Using a 1-cm pathlength of light, the extinction coefficient for nucleotides at 260 nm is 20 per cm per M. Based on this the absorbance at 260 nm in a 1-cm quartz cuvette of a 50 μg/mL solution of double stranded DNA,  33 μg/mL solution of single stranded DNA or a 40 μg/mL solution of  single stranded RNA are all equal to 1 OD.

The extinction coefficients enable DNA measurements without preparing a standard curve. 

Pathlength Correction
With the SPECTROstar^® Nano it is possible to measure in cuvettes and also in microplates. Cuvette based measurements show the advantage that all absorbance values are automatically normalized to 1 cm. These values can be directly used to calculate the DNA concentration using Beer’s law (equation 2) and known extinction coefficients (Table 1).

In microplates the pathlength will vary, depending on the volume of liquid in the well along with the height and dimensions of the well (i.e. 96- versus 384-well plates). To obtain data that can be used in Beer’s law it is necessary to normalize the absorbance results to a 1 cm pathlength (b in equation 2). Pathlength correction can be achieved through a number of methods:

• Use a microplate with a defined pathlength (e.g. LVis Plate, pathlength = 0.5 mm). OD measurements are taken and a standard multiplication value is then  applied to achieve a 1cm pathlength.
• Use of the pathlength correction feature in the software for standard microplates. The volume and microplate used is specified in the test protocol and an algorithm will then be applied to the data. 
• Use a known water peak value correction to normalize the data.

One advantage of using a water peak correction over other methods is that a pathlength can be created for each well allowing different volumes to be dispensed  into one microplate if needed, more importantly this method will also correct for pipetting errors that may  occur during sample preparation.

Purity Determination
DNA samples can contain impurities that will affect the values at 260 nm. Therefore it is recommended to also measure wavelengths where impurities have an  absorbance maximum:

1.     Contamination by protein (280 nm)
2.     Contamination by phenolate, thiocyanate (230 nm)
3.     Scattering of light caused by particulates (340 nm)

A common purity check is to calculate the 260/280 ratio in order to look for protein contaminations. Pure DNA gives a ratio between 1.8 – 2.0, whereas pure RNA shows ~ 2.0. High quality samples should be above 1.7. Another commonly used step is to correct OD values for scattered light by subtracting the OD value at 340 nm from the OD values at 260 or 280 nm, this greatly reduces the standard deviation and increases the sensitivity of measurements.

Materials & Methods

• UV-Cuvettes half semi-micro from Brand 
• UV Star 96-well plate from Greiner 
• Black 384-well LoBase plate μClear, COC with UV bottom from Greiner 
• SPECTROstar Nano equipped with LVis Plate

Herring Sperm dsDNA and HPLC grade water was obtained through normal distribution channels.

Results & Discussion

DNA measurements using the LVis Plate
The LVis Plate is a special microplate that contains 16 sites suitable for measuring 2 μL samples. After pipetting onto the microdrop well on the LVis Plate, the lid is closed resulting in a pathlength of 0.5 mm being generated for all 16 sites. A dsDNA standard curve for measurements with the LVis Plate is shown in Fig. 1.

Sensitivity values (LOD) will depend on how reliably the blank can be measured. The DNA sensitivity for the LVis Plate was calculated to be <2 μg/mL. 

DNA measurements using a standard microplate
In microplates the best sensitivity is achieved when the highest possible volume is used generating a longer pathlength. This is OK generally but if sample is limited then users will usually want to use as low of a volume as possible. Therefore, it is necessary to find the best compromise between volume and sensitivity. Table 2  shows the limit of detection obtained for different  volumes in different microplates. The data presented in Table 2 further indicate that correcting the data at 340 nm generally leads to a higher sensitivity.

Table 2: dsDNA sensitivity in 96-well and 384-well plates.