Guide to Nucleic Acid Purification

DNA and RNA sample preparation is highly honed. What was once the first technique taught to beginning scientists has become an indispensable tool for research and clinical applications. Technology improvements, up-to-date methods, and instrumentation innovation drive global DNA and RNA preparation markets. Unfortunately, demand is out-pacing solutions.

"The bottleneck is in the front end, sample prep. Once you determine those steps, the downstream steps are very similar depending on whether you bind the nucleic acid to silica or magnetic beads," commented Dan Kephart, Ph.D., former Vice President of Research & Development at LGC. "The problem is every mammalian tissue and bodily fluid has its own unique set of hurdles; and don't even get me started on the difficulties of extracting nucleic acids from plants."

Dr. Kephart's sentiment is echoed by Paul Eder, PhD, Senior Scientific Officer at NIAID/NIH, "Most times, we are looking for a virus or microbe whose genome is overshadowed a billion-fold by the host's genomic DNA."

Companies are focusing on faster and more accurate nucleic acid purification products; investments in research and development and the focus on developing COVID-19 vaccines/drugs are two promoting factors. Moreover, demand for more sensitive and specific DNA and RNA sample preparations will ultimately fuel clinical protocols, which rely heavily, either alone or in combination with other diagnostics, on in vitro analyses of specimens to support and/or guide diagnosis and therapeutic monitoring. Below are three queries to help guide researchers through nucleic acid extraction. The main steps in all nucleic acid extractions are presented in the figure below.

1. What are you purifying the nucleic acid from? Each sample type is evaluated based on what specific challenges are likely to be present (protein, paraffin, other nucleic acids). Different samples necessitate different purification strategies. Some samples, such as cultured mammalian cells, are relatively easy to lyse, while fibrous tissue, microbe cell walls, or plant cells require special chemicals, enzymes, or mechanical pressure, to lyse. Disruption is not only important for nucleic acid purification, but also for recovering other constituents of the cell.

There are two approaches to lysing cells and tissues: chemical and mechanical disruption. Osmotic shock (gentle, inexpensive), enzymatic digestion (gentle, inexpensive at small scale), detergents (gentle, moderately expensive), and alkali treatment (harsh, inexpensive), are all examples of chemical approaches to cell or tissue disruption.

These procedures aren't necessarily stand alone; they can be used in combination as well. For example, disruption using osmotic shock can also utilize proteases (proteinase K) to remove protein contaminants or add DNase to samples where the researcher is specifically purifying RNA. By contrast, isolating a low-abundance nucleic acid in a substantial background of another nucleic acid might require a chemistry that will efficiently capture that type of preferred nucleic acid. Although capture technologies are sensitive and efficient, discovery efforts can be thwarted by chromosomal translocations that will not be detected during subsequent PCR-based steps involving amplicons.

In addition to chemical approaches, there are mechanically disruptive forces. These include pestle (or blade) homogenization (moderate, moderately expensive), ultrasonication (harsh, moderately expensive), cavitation (harsh, moderately expensive), "French press" (harsh, moderately expensive), and ball mill (harsh, inexpensive). These forces utilize shearing and tangential forces to produce holes and tears in the cell membrane and/or cell wall, allowing the cellular contents to be collected. Most of the mechanical disruptions create large amounts of heat and should be performed in a cold environment.

Along with these lysing approaches is the inclusion of chaotropic reagents designed to denature nucleases and other contaminating moieties. Such methods can include guanidinium thiocyanate (GuSCN) phenol-chloroform, alkaline extraction, CTAB extraction, Chelex extraction, and ethidium bromide-cesium chloride gradient centrifugation.

GuSCN-phenol-chloroform has the advantage of producing a high yield of high-purity DNA or RNA. It has the disadvantage of hazardous chemical storage and waste. Alkaline extraction is fast and reliable but lacks the level of purity of GuSCN-phenol-chloroform and can fragment genomic DNA so it is usually reserved for plasmid DNA isolation. Cetyltrimethylammonium bromide (CTAB) extraction is mainly used for plant samples and various hard-to-lyse samples, however, the method is time-consuming and requires the use of hazardous chemicals. Chelex extraction is fast but produces nucleic acids of low yield and purity. Finally, cesium chloride gradient centrifugation with ethidium bromide (EtBr) produces high purity and yield of nucleic acids, but is laborious, time-consuming, costly, and EtBr is a the hazardous chemical. Clearly, there are methodological purification options that will depend on the sensitivity and purity of the type of nucleic acid being sought.

2. What type of nucleic acid are you looking for? Clinical applications utilizing cell-free DNA (cfDNA) are swiftly expanding the field of oncology by facilitating early detection of circulating tumor DNA (ctDNA) in different body fluids via liquid biopsy. Moreover, cancer types and specific molecular signatures can be detected in very early stages of tumor development. The increasing number of applications that use cfDNA has fueled the identification and diagnostic utilization of other low abundance nucleic acids, such as cell-free RNAs (cfRNAs), including, but not limited to:

• microRNA (miRNA), small non-coding RNAs of 18–25 base pairs (bp)
• long non-coding RNA (lncRNA), generally greater than 200 bp in size
• circular RNA (circRNA), non-coding RNAs with the size of approximately 2000 bp.

Due to their encapsulation into extracellular vesicles (micro-vesicular bodies (MVBs) or exosomes), their concentrations are relatively stable, albeit in low abundance, in sera, plasma, or other bodily fluids.

3. What do you need the nucleic acid for? The extraction step(s) affect the performance of downstream diagnostic tests with a direct correlation between the efficiency of nucleic acid extraction and the sensitivity of test results. Clinical specimens have distinct and often inhibitory characteristics, reducing the accuracy of PCR or NGS. For example, blood and stool samples contain heme and bile salts that act as inhibitors of PCR-based amplification diagnostics. To circumvent these inhibitors, the extraction method must be controlled by evaluating routine tests of specific DNAs and/or RNAs from these specimens.