DNA quantification methods - Berthold Technologies GmbH & Co.KG

Methods based on absorbance

UV Spectrophotometry using absorbance at 260 nm

Absorbance has been the method of choice for routine quantification of DNA and RNA since decades. It is simple and convenient to use as no further sample treatment (other than DNA extraction) or reaction with other substances is required. However, it is not very specific (it measures all nucleic acids as a whole) and sensitive to contaminants, so it demands very pure DNA to be accurate. Learn more about DNA purity, how to measure it and how it affects quantification.

Absorbance of DNA samples at 260 nm is currently most often measured using a microvolume spectrophotometer, but it is also possible to use a cuvette spectrophotometer or a microplate reader.

Instruments for DNA quantification

In this method, the concentration of a substance is calculated according to the Beer-Lambert law based on its absorbance. The following formula can be obtained from the original formulation of the law:

Where A = absorbance at a given wavelength, ε = extinction coefficient, b = pathlength of the spectrophotometer, c = concentration of the sample.

Hence, for a pathlength of 1 cm, the concentration is equal to the absorbance at 260 nm (the absorption peak of nucleic acids), divided by the extinction coefficient.

Formula to calculate DNA concentration

dsDNA has an extinction coefficient of 0.02 (µg/mL)^-1 cm^-1, hence:

The same formula can be used with the respective extinction coefficients for ssDNA (absorbance x 37 µg/mL) and ssRNA (absorbance x 40 µg/mL). However, it is important to note that the formula is only valid for large nucleic acid molecules with a similar proportion of all nucleotides, such as genomic DNA, plasmids, etc. For oligonucleotides and other short nucleic acid molecules such as miRNAs, the extinction coefficient has to be calculated from the sequence of the oligonucleotide.

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Tristar 5 Multimode Reader

The Tristar 5 is a modular high-performance microplate reader equipped with independent, user-selectable filters and monochromators on both, the excitation and emission side for any measurement

μDrop Plate for low-volume measurements

The μDrop Plate is designed for quick and easy measurement of low sample volumes down to 2 μL. It is an ideal tool for photometric DNA or RNA quantitation and purity analysis

The diphenylamine method (Dische's Test)

Another method of quantifying DNA by absorbance is the diphenylamine method, which is based on the reaction of diphenylamine with deoxyribose sugar to form a blue complex. This method is time-consuming, has a low sensitivity and is therefore no longer used in most laboratories. However, the measurement is made in the visible range and can be performed at 595 nm with a standard ELISA reader if no other instruments are available.

Methods based on fluorescence

The use of fluorescent dyes permits the quantification of DNA with much higher sensitivity than measuring absorbance of DNA itself (typically 10-100 times higher, depending on the specific methods compared). In addition, specific dyes can be used to stain only specific types of nucleic acid, such as dsDNA or RNA, thereby increasing the specificity of the quantification and reducing the effect of contaminants. However, fluorescence-based methods are more expensive than measuring absorbance at 260 nm and often require a standard curve to be prepared. Fluorescence measurement is performed using a microplate reader or a single-tube fluorometer.

Instruments for DNA quantification

There are many different fluorescent dyes for DNA quantification available from a variety of manufacturers. Many of them are offered as kits including dye, DNA standards and dilution buffers. To select a specific kit, it is important to pay attention to the following points:

Specificity of the dye: which nucleic acids can be measured with the dye? Popular options are dyes for dsDNA and RNA, but reagents for ssDNA, miRNA and others are also available.
Detection range: some reagents can be used to detect DNA concentrations as low as 10 pg/uL, but they may lose linearity at higher concentrations. The reagent to be used has to be suitable for the expected concentration range of your samples. This information is usually easily found in the kit insert.
Sample volume requirements: if the sample volume is limited, pay attention to the sample volume required by the kit.
Sample format: some kits have been optimized for measurement in single tubes or cuvettes, while others are intended for use in microplates for higher throughput laboratories. While most DNA quantification kits can easily be scaled up or down easily, selecting a kit that is already optimized for your sample format saves you time and money.
Filters: many common dyes for DNA quantification can be measured with standard fluorescein filters, but before purchasing a reagent, check if your instrument has suitable filters. If you are working close to the detection limit of the kit, you should be prepared to test other filter combinations to achieve the best signal-to-noise ratio.

Methods based on electrophoresis

If the DNA to be quantified is a plasmid, gel electrophoresis is a popular method. In this method, a fluorescent dye such as ethidium bromide or SYBR Green is added to the gel or the samples, followed by electrophoresis of the samples parallel to a DNA ladder and the bands in the gel are visualized with a transilluminator or a gel documentation system. The bands of the DNA ladder have a known mass, so that the amount of DNA in our samples can be estimated by comparing the intensity of their bands with that of the mass ladder. While the method is very specific and ignores most impurities (since the DNA of interest has been separated from other nucleic acids or contaminants by electrophoresis), it is time-consuming, inaccurate (since quantification is performed visually or using image analysis software) and has low sensitivity.

Methods based on PCR

It is also possible to use quantitative real-time PCR (qPCR) and digital PCR or Droplet Digital PCR (ddPCR) to quantify DNA. Digital PCR even enables nucleic acid quantification without a standard curve, since the molecules are separated according to the Poisson distribution. This results in a digital event in each reaction vessel (chip or droplet), so that the proportion of reaction vessels in which amplification has taken place is directly proportional to the amount of DNA sequence used, and thus the amount can be determined directly. The procedure is very sensitive, but requires a special and expensive apparatus as well as a relatively complex experimental setup and more time than the other methods described.

Comparison of DNA quantification Methods

Method	Sensitivity	Advantages	Limitations
UV spectrophotometry	2 ng/µL (typical, with microvolume spectrophotometer)	Simple Fast	Low specificity Sensitive to contaminants
Diphenylamine method	3 µg	Can be measured with colorimeter or ELISA reader	Laborious Time-consuming
Fluorescence measurement	10-50 pg/µL (typical, depending on kit used)	High specificity (nucleic acid type)	Expensive reagents needed
Electrophoresis	10 ng (typical, depending on specific ladder and imaging conditions)	Very high specificity (band of a specific molecular weight)	Time-consuming Low accuracy