Nucleic acid extraction widely encompasses the processes of cell collection, lysis, nucleic acid isolation, purification, and recovery. Samples entering clinical or research laboratories for nucleic acid extraction range from blood and saliva, to fresh, frozen, or decades-old archived tissue biopsies, to aquatic samples and soils of environmental or agricultural interest.
Evaluation of extraction quality is ultimately functional. A successful extraction is one that yields nucleic acid material of sufficient quantity, quality, purity, and integrity that it can be used as substrate material for the intended laboratory protocol.
Manual methods
Manual methods of nucleic acid extraction fall into two broad categories: liquid-phase extractions and solid-support extractions. Though largely replaced by solid-support methods, the liquid-phase phenol/chloroform extraction method is still used and is itself a subject of current research and improvement efforts. The 2018 research paper, “Optimization of phenol/chloroform RNA extraction” describes a modification to the familiar 1987 protocol that reduces contaminants in the final preparation by adding a new chloroform washing step, several RNA washing steps, and two additional ethanol washes. The result, authors of the 2018 paper conclude, is lower threshold values after reverse transcription and RT-qPCR, and greater low-abundance transcript detection sensitivity.
Solid-support extractions are based on reversible associations between nucleic acids and silica matrix column packing, or magnetic beads functionalized with surface chemistries specific for the nucleic acid species of interest. In both cases, the process is to bind nucleic acids to the solid support in a buffer that favors the association, wash away contaminants, and elute the isolated nucleic acid into a small volume of buffer. An advantage of the magnetic-bead method is the absence of forceful column washing, frequently driven by column centrifugation, which can shear long molecules.
Automation
Nucleic acid extraction automation is largely aimed at replacing manual benchtop processes such as buffer exchanges, sample tube movement between racks and stations, microplate stacking, etc. with robotic systems. Advantages of automation include consistent protocol repetition and hands-off time for lab personnel to attend to other tasks. Automated systems for nucleic acid extraction automation fall into two categories: multiple-purpose automation platforms and systems dedicated to nucleic acid extraction.
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Many makers of multi-functional benchtop robotic systems have worked out hardware control software that automates protocols found in the user guides of nucleic acid extraction kits. In addition to the general advantages of automation, these versatile systems allow the possibility of integrating a nucleic acid extraction protocol with the downstream application for which the extracted material is intended. Referring to the company’s benchtop automation product, Bruce Jamieson of Hudson Robotics said, “The SOLO system supports extracted DNA normalization with an OD plate reader. We can attach a thermal cycler on the deck and do PCR. We’ve automated full NGS library prep processes, and we just competed a successful validation of the Lucigen NGS kits.”
Dedicated devices for nucleic acid extraction take up less space and are tightly linked and optimized for use with a limited number of kits. Makers expand the capabilities of these systems by developing compatible new kits and protocols. Recently, Promega introduced the Stabilized Saliva DNA kit for use with their Maxwell RSC system, and QIAGEN just introduced the DNeasy PowerSoil Pro Kit, which is compatible with their QIAcube system.
Special applications
Typically, cell collection and lysis are carried out as immediately prior to the purification steps detailed above as possible, in order to minimize loss of nucleic acid yield or quality due to degradation. Two notable exceptions are formalin-fixed paraffin-embedded (FFPE) samples and blood collection cards.
Worldwide archives of FFPE tissues with associated clinical histories represent a highly valuable source of material for biomedical research. However, the formalin fixation process, originally conceived to preserve microscopic detail at the multi-cellular level, is damaging to nucleic acids. The process of nucleic acid extraction is further complicated by the requirement of de-paraffinizing samples to release cells for analysis.
Kits for nucleic acid extraction from FFPE samples represent an active area of product development, which will ultimately enable large retrospective studies. Recently, a research group conducted a rigorous comparison of seven commercial kits for the extraction of nucleic acids from FFPE samples. While all but one of the kits tested were of the “spin-column” variety, there were greater differences among the kits relative to de-paraffinization methods, functional capture surfaces, and buffer compositions. The study, published this year in Plos One, found “variable quantity, quality, and performance of the isolated nucleic acids extracted by the kits tested. In the open-access article, the authors provided useful information to enable researchers to select the “optimal procedure” for a given sample type and intended downstream application.
Blood collection cards are composed of plain filter paper, or the same paper treated with a chemical mixture for biomolecule preservation. Preserved blood samples spotted onto the cards can be stored for lengthy periods of time, or transported at ambient temperature, until the extraction process is performed.
Though blood collection cards have existed for decades, global health concerns have re-ignited product R&D. “One of the things I’m seeing a strong increase in is nucleic acid extraction from paper, and the dominant driver of this is HIV and AIDS,” said James Nelson, Ph.D., of GenTegra LLC, a maker of ambient-temperature storage products for biologicals.
Routine HIV RNA testing is used to determine viral count and monitor the progression of AIDS. Aggregate data are used to evaluate the success of retroviral therapies in purging viral reservoirs from at-risk populations.
“There are 1.8 million new cases of AIDS each year, and 70% of those living with AIDS are in Africa, where cold-chain transport is frequently not an option,” explained Dr. Nelson. “But in post-marketing studies this year, we discovered that our blood collection cards release 95% of the nucleic acid material from dried blood spot samples. Conventional filter-paper based cards give up only about half. This difference in extraction yield increases the efficiency of HIV surveillance and will have an important effect on efforts to eradicate the virus.”
A novel approach
Representing a departure from previous extraction methods, a new technology under development at Purigen Biosystems uses the principle of ITP (isotachophoresis) to separate DNA from cell lysates and to concentrate the purified DNA in a small buffer volume.
ITP takes advantage of the characteristic velocities of charged molecular species when they are exposed in solution to an electric field. Unlike gel electrophoresis techniques , ITP separates and focuses charged molecules based on their ionic mobilities rather than size.
In an electric field, a "leading," electrolyte containing high-mobility ions migrates rapidly and maintains separation from a "trailing" electrolyte containing lower-mobility ions. When cell lysate is introduced, its constituent DNA migrates more rapidly than the trailing electrolyte, but more slowly than the leading electrolyte, becoming purified and concentrated at a focal point from which it may be recovered.
"Isotachophoresis is a novel approach to extracting and recovering DNA from cells," said Barney Saunders, Ph.D., of Purigen Biosystems. “Despite protocol automation and other advances, conventional extraction methods are affected by variable yields and purities, complex protocols, salt and solvent carryovers, and waste-stream issues. In ITP, the salt concentrations are much lower and there are no solvents or detergents other than what you might use to lyse your cells. There's no binding, washing, or centrifugation. Actually, no moving parts are involved," he added.
Purigen Biosystems intends to make their isotachophoresis technology available for testing through an early access program.