Current Nucleic Acid Extraction Methods

Current Nucleic Acid Extraction Methods

PART 1

Nucleic acid extraction (NAE) is one of the most pivotal steps in molecular biology, being routinely used in many areas of the biological and medical sciences, as this procedure marks a starting point in any molecular diagnostic kit [1]. This crucial procedure has been known for over a century and has developed substantially over the last decades. However, some progress still has to be achieved so that NAE protocols leave the laboratory settings into the “real world” of point-of-care diagnostics (POC-Dx).

Nowadays, it is known that intracellular nucleic acids (NAs) may be broadly categorized as genomic (or chromosomal), plasmids, and different types of RNAs . Although RNAs possess uracil while DNAs present thymine , nucleic acids exhibit similar basic biochemical properties but might have quite distinct tridimensional structures (genomic, plasmid, tRNA, mRNA, rRNA, etc). However, despite the structural differences, the most commonly used methods described in the present text can be applied to DNA in its many organizational formats (chromosomal, plasmid, etc.), as well as RNA and its multidimensional formats (mRNA, rRNA, tRNA, miRNA, etc.) with minor modifications.

NAE can be roughly divided into four steps, which can be modulated depending on the sample and downstream applications: (i) cell disruption; (ii) removal of membrane lipids, proteins, and other nucleic acids, (iii) nucleic acid purification/binding from bulk; and (iv) nucleic acid concentration .

Cell disruption or disintegration can be achieved by physical and/or chemical methods, whose main aim is to disrupt the cell wall and/or cellular membranes. Disruption methods are mainly based on properties of the sample and for this purpose a wide range of tools and approaches are used either alone or combined to achieve tissue/cell disruption . Lytic enzymes, chaotropic agents, and different types of detergents are the main components of chemical lysis, while mechanical method disrupts the cells by grinding, shearing, bead beating, and shocking . It is interesting to note that if one technique does not yield good results, another might prove successful. Osmotic shock methods have yielded, in certain cases, better results than common NA purifications protocols such as phenol-chloroform extraction and bead beating . Not only is cell disruption important for DNA extraction, but it also plays a crucial role in the biopharmaceutical industry, as many recombinant proteins and other important constituents of the cell can be recovered through this process. Another approach for cell disruption is the use of different methods in combination. A good example is the case for enzymatic lysis, where many protocols use proteases to free the NA from its protective protein scaffold. Also, the inactivation of cellular nucleases that come free into solution in order to protect the new protein-free NA is crucial. A combination of detergents and chaotropic salts in a single solution is used to solubilize cell wall and or cell membrane and inactivate intracellular nucleases . Mechanical disruption, on the other hand, makes use of force to extract out constituents of the cell. A classic example of grinding in biosciences is the use of mortar and pestle, which is nowadays optimized with the use of liquid nitrogen (when allowed by the sample). Cells walls can also be disrupted by the shock waves created by rapid changes in pressure elicited by sonication or cavitation . Other mechanical tools available for cell disruption are shearing, which use a tangential force to make a hole in the cell, and bead beating, which uses different glass or steel beads to rupture tough cell wall as mentioned.

NAE methods encompass extraction of both DNA and RNA but can be more broadly characterized into chemically driven or solid-phase methods; both contain the four steps mentioned above . In the next sections, we will review the working principle of and/or rationale for the main methods used nowadays in the biological and medical sciences. Since molecular diagnostics rely heavily on techniques that start with NAE, we will also discuss some of the basic features of devices available for POC-Dx, culminating with the challenges and limitations of adapting NAE methods to point-of-care diagnostic tests.