Why Spectrophotometry Still Rules Nucleic Acid Quantification

Nucleic acid (NA) quantification is arguably the most important assay in a molecular biology lab. It serves as the gatekeeper for everything that follows downstream.

Micro volume UV-vis spectrophotometry is indisputably the preferred method of quantifying NAs due to a large number of factors including ease and simplicity of the assay, and ubiquity of the equipment, with an estimated saturation rate of over 90% of labs worldwide owning or having access to an instrument. There are nonetheless a variety of ways that researchers quantify NAs, each with its own distinct advantages and drawbacks, which serve mostly as orthogonal or complementary techniques for verification purposes. Here we explore why spectrophotometry is such a popular method for quantifying nucleic acids, with an eye toward helping researchers understand when spectrophotometry is the right tool for their workflow compared to alternative quantification methods, and when it should be supplemented.

Why quantify?

It’s hard to imagine a molecular biology protocol that doesn’t call for a specific amount or concentration of nucleic acid to be used. Whether for CRISPR workflows, next-generation sequencing, restriction digestion, ligation, or genotyping, quantifying the target material is a requisite step in the protocol. For qPCR, for example, demonstrating nucleic acid quantity and amount is considered an essential part of transparent experiment reporting in MIQE guidance.

Sure, for a quick experiment, spectrophotometric quantification can give an on-the-fly first impression of how much DNA to add to an experiment.

Yet quantification “is not just a yield measurement,” notes Mariya Hrynchak, Product Portfolio Manager for Nippon Genetics. “It is a checkpoint that helps scientists decide whether their sample is concentrated enough, clean enough, intact enough, and comparable enough to trust the downstream result.”

History of UV-vis and how and why microvolume spectrophotometry came to dominate

Before the turn of the millennium, researchers would generally quantify their DNA and RNA by placing several microliters of sample into a one-centimeter cuvette, and place that cuvette into a bulky benchtop spectrophotometer, to take optical density (absorbance) measurements at several wavelengths in the ultraviolet and visible (UV-vis) ranges. Samples were often too concentrated to be read by a traditional spectrophotometer and so needed to be diluted, sometimes serially, down to an operative absorbance range. Readings were then compared to those standards. The process was wasteful of both sample and reagents, time-consuming, and error prone.

In the early 2000s, largely coinciding with the era of “molecular biology exploding into the market,” there was a desperate need for a very quick method for quantification that didn’t take up a lot of sample, recalls Andrew Jones, Market Development Manager, DeNovix.

Enter the method of micro-volume spectrophotometry. It requires only one microliter of sample. It’s very quick, taking “just a few seconds to measure a sample. You don’t need a set of standards—all you need is a blank, so whatever your sample is suspended in,” and you don’t have to spend the time or money preparing an additional assay, he explains.

Now the technology is very mature, and the instrumentation is quite ubiquitous. “Generally speaking people have access to this technology, and they have access to people who understand this technology,” Jones says. Very little training is necessary for a regular lab member—the training with UV-vis is often more about understanding the limitations of the method when dealing with sub-optimal samples, for example, than general operational procedures.

The ratios, and beyond

In addition to quantification, spectrophotometers can also provide information about contamination in the sample.

NA quantification is basically a measure of how much light the nucleic acid absorbs, with concentration proportional to the absorption at a specific wavelength. Pure DNA and pure RNA, for example, absorb maximally at 260 nm. “Anything absorbing at or near that wavelength can affect the results,” notes Hrynchak.

Contaminants such as oligonucleotides, phenol, and coenzymes like NAD+, for example, also contribute to absorption of 260 nm light, but differentially absorb at other wavelengths. Researchers will often look at and report the 260/280 ratio as a measure of protein contamination, and likewise the 260/230 ratio to indicate the presence of, for example, phenol and chaotropic salts. “The two ratios give you a nice impression of contamination in your sample, as well as getting a quantification of 260 nm for nucleic acids,” Jones points out.

Alternatives?

Not all the information a protocol may call for can be obtained by UV-vis spectrophotometry. Many other techniques can substitute for, or complement, UV-vis spectrophotometry for determining how much DNA or RNA is in a solution—some being familiar tried-and-true methods for other tasks, others less so for any given lab.

Take electrophoresis, for example. It requires standards and is much slower than spectrophotometry, but yields information about fragment size, degradation, and integrity.

If the key question is how much amplifiable target is present—especially in degraded, forensic, clinical, or FFPE samples—qPCR or digital PCR may be the way to go, though both are time-consuming.

Low-concentration samples may also benefit from the specificity and sensitivity of fluorometry. Fluorometry is simple and requires only a relatively inexpensive assay, which allows it to distinguish RNA from single-stranded DNA from double-stranded DNA. It excels at quantification of total DNA, but by itself gives no information about contamination.

All (or some) together now

No single technique can assess all of the parameters that a researcher may want to know about a nucleic acid solution.

Hrynchak sums it up this way:

• Spectrophotometry asks “how much UV-absorbing nucleic acid material is there, and how clean does it look?”
• Fluorometry asks “how much of this nucleic acid class is present?”
• Electrophoresis asks “what size and integrity does it have?”
• And qPCR or digital PCR asks “how much usable target is present?”

The bottom line? Most researchers rely on the spectrophotometer for a quick-and-dirty quantification, and when they need to, use a combination of techniques to get a fuller picture of their solution.