DNA Quantification Methods and Applications

DNA quantification is the process of measuring the concentration and purity of DNA in a sample, typically expressed in ng/µL or µg/mL. It is crucial to select the right method for DNA quantification to ensure optimal results in your downstream molecular biology applications. Commonly used methods for DNA quantification include spectrophotometry, fluorometry, agarose gel electrophoresis and real-time PCR (qPCR).

The right instrument for DNA quantification depends on the type of technology you use for DNA quantification.

If you perform agarose gel analysis of DNA fragments, you can increase throughput, speed and resolution with the QIAxcel Connect and QIAxcel DNA Kits. QIAxcel technology provides fully automated size separation and quantification of DNA fragments in up to 96 samples per run. The capillary-electrophoresis instrument simplifies analysis by using ready-to-run gel cartridges that eliminate tedious gel preparation. Automated analysis reduces hands-on time and manual handling errors.

If you work with forensic DNA samples, qPCR is the method of choice due to its high sensitivity and specificity with results in real time. The Rotor-Gene Q and Investigator Quantiplex Kits provide superior sensitivity and reaction speed. They are designed to establish whether a sample contains sufficient and intact human DNA to enable STR or SNP analysis by capillary electrophoresis or next-generation sequencing (NGS).

Each method for DNA quantification has advantages and disadvantages. The choice of method depends on factors such as sample type, experimental need and your downstream application. Make sure to work according to DNA best practices. Here’s a summary of the available methods:

• Spectrophotometry: Measures microgram quantities of DNA
• Fluorometry: Measures nanogram DNA quantities
• Agarose gel electrophoresis: Quantifies small DNA fragments
• Real-time PCR (qPCR): Quantifies DNA samples and forensic DNA samples

DNA concentration can be determined by measuring the absorbance at 260 nm (A260) in a DNA spectrophotometer using a quartz cuvette. For greatest accuracy, readings should be between 0.1 and 1.0. An absorbance of 1 unit at 260 nm corresponds to 50 µg genomic DNA per mL (A260 =1 for 50 µg/mL; based on a standard 1 cm path length. This relation is valid only for measurements made at neutral pH, therefore, samples should be diluted in a low-salt buffer with neutral pH (e.g., Tris·Cl, pH 7.0). For an example of the calculation involved in nucleic acid quantification when using a spectrophotometer, see Spectrophotometric measurement of DNA concentration.

When working with small amounts of DNA, such as purified PCR products or DNA fragments extracted from agarose gels, quantification through agarose gel electrophoresis analysis may be more effective.

Tip: If you use more than one cuvette to measure multiple samples, the cuvettes must be matched.

Tip: Spectrophotometric measurements do not differentiate between DNA and RNA, so RNA contamination can lead to overestimation of DNA concentration.

Tip: Phenol has an absorbance maximum of 270–275 nm, which is close to that of DNA. Phenol contamination mimics both higher yields and higher purity, because of an upward shift in the A₂₆₀ value.

Effects of solvents on spectrophotometric readings

Absorption of nucleic acids depends on the solvent used to dissolve the nucleic acid (7). A₂₆₀ values are reproducible when using low-salt buffer, but not when using water. This is most likely due to differences in the pH of the water caused by the solvation of CO₂ from air. A₂₆₀/A₂₈₀ ratios measured in water also give rise to a high variability between readings (see figure Effect of solvent on A₂₆₀/A₂₈₀ ratio) and the ratios obtained are typically <1.8, resulting in reduced sensitivity to protein contamination (7). In contrast, A₂₆₀/A₂₈₀ ratios measured in a low-salt buffer with slightly alkaline pH are generally reproducible.

Effect of RNA contamination on spectrophotometric readings

Depending on the sample disruption and lysis method and the DNA extraction and purification kits used, RNA may be co-purified with genomic DNA. RNA may inhibit some downstream applications, but it will not inhibit PCR. Spectrophotometric measurements do not differentiate between DNA and RNA, so RNA contamination can lead to overestimation of DNA concentration. RNA contamination can sometimes be detected by agarose gel analysis with routine ethidium bromide staining, although not quantified effectively. RNA bands appear faint and smeary and are only detected in amounts ≥25–30 ng (0.5:1 RNA:DNA ratio).

Treatment with RNase A will remove contaminating RNA; this can either be incorporated into the purification procedure or performed after the DNA has been purified. Prior to use, ensure that the RNase A solution has been heat-treated to destroy any contaminating DNase activity. Alternatively, use DNase-free RNase purchased from a reliable supplier.

RNA contamination of plasmid DNA can be a concern depending on the method used for plasmid preparation. Methods using alkaline lysis with phenol extraction cannot separate RNA from plasmid DNA, leading to high levels of RNA contamination. Advanced plasmid anion-exchange technology allows isolation of high-molecular-weight genomic DNA that is free of RNA.

DNA purity

The ratio of the readings at 260 nm and 280 nm (A₂₆₀/A₂₈₀) provides an estimate of DNA purity with respect to contaminants that absorb UV light, such as protein. The A₂₆₀/A₂₈₀ ratio is influenced considerably by pH. Since water is not buffered, the pH and the resulting A₂₆₀/A₂₈₀ ratio can vary greatly. Lower pH results in a lower A₂₆₀/A₂₈₀ ratio and reduced sensitivity to protein contamination (7). For accurate A₂₆₀/A₂₈₀ values, we recommend measuring absorbance in a slightly alkaline buffer (e.g., 10 mM Tris·Cl, pH 7.5). Be sure to zero the spectrophotometer with the appropriate buffer.

Determining DNA purity by absorbance at 260, 280 and 320 nm

Pure DNA has an A₂₆₀/A₂₈₀ ratio of 1.7–1.9. Scanning the absorbance from 220–320 nm will show whether there are contaminants affecting absorbance at 260 nm. Absorbance scans should show a peak at 260 nm and an overall smooth shape.

Fluorometry

Fluorometry allows specific and sensitive measurement of DNA concentration by using fluorescent dye, with common dyes including Hoechst dyes and PicoGreen.

Hoechst 33258 has little affinity for RNA, allowing accurate quantification of DNA samples that are contaminated with RNA. The dye shows increased emission at 458 nm when bound to DNA. DNA standards and samples are mixed with Hoechst 33258 and measured in glass or acrylic cuvettes using a scanning fluorescence spectrophotometer or a dedicated filter fluorometer set at an excitation wavelength of 365 nm and an emission wavelength of 460 nm. The sample measurements are then compared to the standards to determine DNA concentration.

Tip: As Hoechst 33258 preferentially binds AT-rich DNA, use standards with a similar base composition to the sample DNA.

PicoGreen is a highly sensitive measure of dsDNA and can measure as little as 20 pg dsDNA in a 200 µL assay volume. Indeed, DNA concentrations from 500 pg/mL to 500 ng/mL can all be measures using a single dye concentration. The assay is optimized to minimize the fluorescence contributions of RNA and ssDNA, such that dsDNA can be accurately quantified in the presence of equimolar concentrations of ssDNA and RNA with minimal effect on the quantitative results.

Agarose gel analysis

Agarose gel analysis enables quick and easy quantification of DNA, especially for small DNA fragments (such as PCR products). As little as 20 ng DNA can be detected by agarose gel electrophoresis with ethidium bromide staining. The DNA sample is run on an agarose gel alongside known amounts of DNA of the same or a similar size. The amount of sample DNA loaded can be estimated by comparison of the band intensity with the standards either visually or using a scanner or imaging system. Be sure to use standards of roughly the same size as the fragment of interest to ensure reliable estimation of the DNA quantity, since large fragments interchelate more dye than small fragments and give a greater band intensity.

More precise agarose gel quantification can be achieved by densitometric measurement of band intensity and comparison with a standard curve generated using DNA of a known concentration. In most experiments the effective range for comparative densitometric quantification is between 20 and 100 ng.

Tip: The amount of DNA used for densitometric quantification should fall within the linear range of the standard curve.

See DNA analysis using analytical gels for further information on agarose gel electrophoresis.

Real-time PCR

In forensic sample analysis, quantification of the human DNA in a sample confirms that the sample contains sufficient human DNA for further testing, establishes whether it contains inhibitors and predicts the success of your STR analyses so you can adjust your downstream assay strategy accordingly. The most commonly used method for quantification of human DNA is real-time PCR (qPCR). Learn more about qPCR in human DNA quantification.

Discover how to maximize your PCR success.

DNA quantification measures the amount and concentration of extracted DNA in a sample. A variety of methods can be used for this, including spectrophotometry, fluorometry, agarose gel electrophoresis and qPCR.

DNA purity can be determined using DNA spectrophotometry. For accurate A₂₆₀/A₂₈₀ values, we recommend measuring absorbance in a slightly alkaline buffer (e.g., 10 mM Tris·Cl, pH 7.5). Pure DNA has an A₂₆₀/A₂₈₀ ratio of 1.7–1.9. Scanning the absorbance from 220–320 nm will show whether there are contaminants affecting absorbance at 260 nm.

You can estimate DNA concentration by comparing the intensity of the sample’s band to a DNA ladder of known concentration. For more precise quantification, use a spectrophotometer or fluorometer.