Nucleic Acid Isolation and Downstream Applications
What is Nucleic acid?
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). If the sugar is ribose, the polymer is RNA; if the sugar is the ribose derivative deoxyribose, the polymer is DNA.
Nucleic acid isolation and downstream applications
The specific properties of nucleic acids have been widely employed in the development of different molecular methods and mathematical models for their analysis. These methods are applied to identify microorganisms and genetic predispositions, to detect different mutations and determine their role in antibiotic resistance, to study phylogenetic relationships, and so on. What all these methods share in common is their starting point: obtaining a purified nucleic acid sample.
Nucleic acid extraction methods
Since nucleic acid extraction is a starting point in a vast array of downstream applications, the high quality of nucleic acids in the starting samples is a key factor for the success of the subsequent steps of analysis. Thus, nucleic acid extraction could be defined as a series of steps to obtain nucleic acid samples/materials of particular purity that are free of impurities and are suitable for different downstream application steps. The purpose of nucleic acid extraction methods is to disintegrate the cell envelope and achieve maximum elimination of lipids and proteins to obtain pure DNA and/or RNA. This is principally based on heat adsorption on silica membranes/beads, anion exchange chromatography, sedimentation/precipitation, and use of magnetic particles.
The choice of method – in view of optimal time/quality balance – depends on the aim of the study, the type of analysis, the type of nucleic acid, and the cost. It is important to provide appropriate conditions for nucleic acid extraction in order to avoid nucleic acid degradation due to oxidation by reactive oxygen species generated during respiration in vivo or, extracellularly, by mechanisms involving metal ions. Nucleic acid degradation can result from hydrolysis of the 3′-5′ phosphodiester bonds catalyzed by metal complexes as well as from the spontaneous breakage of these bonds due to transesterification via a nucleophilic attack at the phosphorus atom by an adjacent 2′-hydroxyl group.
Two keys of extracted nucleic acids
The quality of the extracted nucleic acids also depends on the quality of the starting sample. In fact, all manufacturers of nucleic acid isolation kits recommend that fresh starting material be used. Another key step is cell lysis, which – if incomplete – would result in reduced yield and column blocking and, in turn, in lower purity.
Geneture Medical provides one stop solution of Nucleic Acid Extraction and Analysis, and has now developed a number of commercial bead-based nucleic acid isolation kits. Our protocol requires fewer steps and reagents than other DNA extraction protocols and can be automated in 24, 96-deep well plates. We know what it takes to succeed. We first work with you to understand your specific challenges and then our experts will design, develop, manufacture, and validate complete assay solutions for your molecular diagnostic assay. Our quality system is an integral part of every step in the molecular diagnostics assay development and manufacturing process. We operate under the most rigorous requirements of ISO 13485:2016, from early-stage development to tech transfer, scale up, validation, verification, and commercial production.
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