Methods for Quantifying Nucleic Acids

Reliable nucleic acid quantification is essential for the majority of downstream research applications. Constantine Garagounis, Product Development & Marketing Specialist at PCR Biosystems, explained that everything from how much DNA template to use in a PCR to the effective library loading in next-generation sequencing (NGS) workflows relies on accurate quantification. “Getting the amount right can mean the difference between a successful experiment or a waste of time and reagents,” Garagounis noted.

Paraj Mandrekar, Technical Services Scientist at Promega, further emphasized the importance of adhering to protocol-specific concentration ranges. “PCR-based processes, especially multiplexed PCR, can be very sensitive to the input amounts of DNA,” he stated. Excessive DNA in multiplex PCR can inhibit the reaction or favor smaller amplicons and more abundant targets, potentially causing rare targets to be missed. Furthermore, insufficient DNA may yield low signal or lost information, which also impacts downstream assays. While modern amplification systems allow downstream assays to accept a wider range of nucleic acid concentrations, Mandrekar noted that amplifying DNA within the correct range is still necessary for success.

Quantification tools and techniques

Ranging from semi-quantitative and simple techniques, like agarose gels or UV wavelengths, to much more complicated quantification via qPCR and NGS, Garagounis explained that there are numerous tools designed for nucleic acid quantification. He noted that each method comes with distinct advantages and disadvantages.

Agarose gels

Using agarose gels for nucleic acid quantification is an affordable and traditional strategy. “Agarose gels, stained with ethidium bromide or other safer alternatives, can provide a rough estimate of how much nucleic acid is present in a sample,” stated Garagounis. The brightness of the DNA bands and the weight/thickness of those bands can be compared to another sample with a known quantity to give a rough estimate of the amount of DNA. While this approach isn’t the most accurate, it’s straightforward, especially if the workflow already involves running the sample on a gel. Garagounis pointed out that this method also provides information on the quality of the sample such as multiple DNA/RNA species or whether the sample is degraded.

Spectrophotometric methods

Another early technique to quantify nucleic acids, explained Mandrekar, was to measure the absorbance of nucleic acids at a wavelength of 260 nm. This spectrophotometric approach is currently best represented in the Nanodrop instrument. Mandrekar also noted that these instruments “can provide an estimate of DNA concentration and simultaneously provide an estimate of the sample purity, specifically through absorbance ratios as 260:280 nm and 260:230 nm.” In the past, spectrophotometers were bulky and required large volumes of samples, but newer devices are fast and can provide a good estimate of yield with minimal volumes (1–2 μL). The downside to this method is that absorbance-based measurements can detect contaminants like nucleotides, proteins, or fragmented DNA that may linger after nucleic acid purification, leading to an overestimation of nucleic acid.

Fluorescence-based quantification

Fluorescence methods for nucleic acid quantification utilize nucleic acid-binding or intercalating dyes. “This technology relies on the ability of some small fluorescent molecules to bind DNA or RNA and increase their fluorescence intensity when bound to a target molecule,” stated Garagounis. Samples mixed with these dyes are assessed in devices like fluorometers or fluorescence plate readers against a reference curve from standard samples.

Mandrekar noted that several commercial systems employ these dyes. These systems are quick, fairly inexpensive, and adaptable to various devices like plate-based readers or handheld fluorometers. However, fluorescent dyes typically focus on double-stranded DNA (dsDNA) and might underreport total DNA by inadequately measuring single-stranded DNA (ssDNA). This method's reliance on the physical state of DNA can lead to inaccuracies, particularly when DNA is in a single-stranded form due to heat or the presence of chaotropic reagents.

PCR-based methods

“Newer techniques tend to incorporate PCR-based measurements,” explained Mandreka. This includes quantitative real-time PCR (qPCR), digital PCR (dPCR), and droplet digital PCR (ddPCR). Mandreka detailed that among these methods, qPCR offers a broad measurement range and can target specific sequences and measure multiple targets in the same sample through multiplexing, but it requires standards for calibration. In contrast, dPCR and ddPCR partitioned reactions provide absolute quantitation without a standard curve and can detect lower amounts of DNA effectively. These methods are often more costly and time-consuming; however, they offer advantages such as multiplex capabilities and reduced impact from inhibitors through partitioning, which can dilute inhibitors to subinhibitory levels.

Selecting a quantification method

With a growing number of quantification methods, choosing the right approach has become more complex, requiring a careful evaluation of the sample properties and the specific demands of the downstream applications. Sample characteristics, such as purity, presence of contaminants, and extraction source, can significantly impact the choice of nucleic acid quantification method. For example, samples with degraded or improperly extracted nucleic acids may yield inaccurately high readings using spectrophotometry or fluorimetry, while those containing PCR inhibitors might not be suitable for qPCR. Garagounis also recommended that researchers with high-value or scarce samples should avoid wasteful methods like gel electrophoresis. Conversely, for precise quantification of specific nucleic acids in complex samples, PCR-based methods are preferable.

Garagounis shared that his personal approach is finding a quantification method that is faster, cheaper, and the least laborious. “If you’re running ligations, restriction digests, or even PCRs, then a rough estimate is enough,” he suggested. In that case, “gel comparisons or photometric data is probably the most sensible approach. If you’re preparing to sequence an NGS library, at a minimum I would recommend a fluorimetric approach or, better yet, absolute quantification via qPCR.”

In addition to these recommendations, Mandrekar stressed finding the quantitation method that best mimics the intended downstream assay. “For example, a PCR-based genotyping method would benefit from having DNA or RNA quantitated by a PCR-based quantitation method.” Mandrekar also shared that various companies, including Promega, offer numerous approaches to quantitation and are typically able to recommend the best methods based on their experience with different purification and quantitation techniques. Technical services at these companies often include teams of specialists in nucleic acid purification and qPCR-based quantitation who are available to assist researchers in making informed decisions tailored to their specific sample types and purification methods.

Finally, Garagounis explained that “researchers should always choose an appropriate quantification technique for their samples, balancing the need for accurate measurement of nucleic acid quantity in their downstream workflow, with sample constraints (quantity and value), the cost and efficiency of the quantification method, and the overall number of samples they need to process.”