Last Updated on
February 8, 2023
DNA purification is a vital procedure in molecular biology and biotechnology labs to determine the success and failure of your workflow. Impurities in DNA samples can affect downstream applications in your lab.
Cleaning up DNA is essential for two reasons:
What does cleaning up DNA mean?
DNA purification or “cleaning up” DNA molecules means removing enzymes, buffers, proteins, lipids, RNA, and other compounds and chemicals that can affect downstream applications.
Working with cell lysates that have these impurities, will not only produce erroneous results at one stage but at all subsequent stages. Thus, by purifying your DNA sample, you reduce such probability and maintain the purity and quality of your nucleic acid.
Especially while performing high-throughput assays in your lab, such as multiplex or real-time PCR, cloning, DNA sequencing, quantitative PCR (qPCR), radioactive and fluorescent sequencing, and next-generation sequencing (NGS), you need high-quality DNA to obtain quality, accurate, and reliable results.
In this article, we will cover the types of DNA purification methods, the procedure to perform the technique, and its applications in biotech and life sciences lab workflows.
Plasmid and genomic DNA samples can be purified using several techniques. Some of these are:
Though it’s an easy-to-perform and cheap approach, this technique is time-consuming and involves the use of hazardous organic solvents.
Some other techniques of performing DNA purification include salting out with potassium acetate or another suitable kosmotrope, use of organic solvents and chaotropes (guanidium salts) for DNA extraction, Cesium chloride (CsCl) purification, affinity techniques using triple helix affinity resins, and Hydroxyapatite-based strategies.
Here’re the basic steps used in DNA purification:
Cell lysates (or other sample types, such as whole blood) are prepared by disrupting cells and releasing their nucleic acid content into the solution. This is done by using three techniques:
This involves cleaning up the cell lysate based on the starting material to remove cellular debris. The step is essential to minimize the carryover of unwanted substances (such as lipids, proteins, and saccharides) into the further reaction. The most common methods of clearing include filtration, centrifugation, or bead-based techniques.
After obtaining purified cell lysate, a range of techniques are used to purify DNA, such as silica, cellulose, ion exchange, or precipitation-based methods.
After the cell lysate is purified, DNA is isolated using a variety of techniques. It includes sample lysis by detergents followed by the purification of the isolated DNA using cellulose, silica, or ion exchange matrices for DNA binding.
Each technique influences the binding capacity of DNA and the efficiency and purity of the isolation. By manipulating binding conditions you can enrich DNA fragments in your sample.
Four commonly used techniques for DNA isolation include:
Many commercial suppliers offer DNA extraction kits and DNA purification kits to perform the isolation of high molecular weight DNA after workflows like agarose gel electrophoresis, Polymerase Chain Reaction (PCR) amplification, and other enzymatic reactions.
The washing step includes removing salts, proteins, and other contaminants using a wash buffer — consisting of reagents like 80% ethanol, 20 mM NaCl, and 2 mM Tris-HCl.
To elute DNA fragments from the matrix, low-ionic-strength solutions, like nuclease-free water and TE buffer are used. However, before deciding on the elution buffer, decide on the purpose of your downstream applications. For example, TE buffer can be useful for eluting and storing DNA if EDTA does not affect downstream processes.
DNA purification has extensive applications in a range of life sciences and biotech industries for its uses in high-throughput workflows, such as PCR, qPCR, DNA sequencing, and many others.
If working on developing transgenic plants or animals, you require pure DNA samples. Furthermore, they also have profound applications in developing DNA vaccines using recombinant DNA.
DNA extraction and purification are necessary to identify and diagnose genetic conditions, such as Huntington’s disease, cystic fibrosis, and Down syndrome. Further, it also has wide application in paternity testing.
You must already know that DNA is the key in forensic labs. Thus, the forensic team collects biological samples, such as hair, blood, saliva, or skin, from the crime scene to obtain DNA, which helps in identifying the suspects.
However, the isolated DNA can be mixed with contaminants, which need to be removed to obtain purified DNA for the right identification. And, that’s why DNA purification is an essential procedure in such labs.
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DNA purification is a powerful tool used in life sciences and biotech labs to extract pure DNA for high-throughput workflows. Any impurities or contaminants in the DNA can affect the downstream applications and determine the result of your research.
Today, a range of techniques are available to clean DNA, which include organic extraction, silica-column-based techniques, ethanol precipitation, and anion-exchange chromatography. The basic steps involved in the procedure are lysate preparation and purification, binding of DNA to the matrix, washing, and elution.
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