DNA Purification: Overview & Applications

DNA Purification: Overview

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:

• Ensure that you obtain a sufficient amount of your genomic and/or plasmid DNA sample when the source is limited to meet your research requirements.
• Reduce the number of contaminants that can compromise your research results. Further, the impurities can also reduce the shelf-life of your precious samples.

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.

Techniques Used In DNA Purification

Plasmid and genomic DNA samples can be purified using several techniques. Some of these are:

• Organic extraction: One of the most traditional techniques used to purify DNA. It’s also known as the phenol-chloroform extraction procedure. The steps followed in this DNA extraction method include:

• Cell lysis, removing cell debris through centrifugation, and obtaining nucleic acids in the supernatant.
• Denaturation of proteins followed by their precipitation using a 1:1 mixture of phenol and chloroform.
• Recovery of purified DNA by precipitating it using ethanol or isopropanol.
• Degradation of RNA using RNase enzyme.

Though it’s an easy-to-perform and cheap approach, this technique is time-consuming and involves the use of hazardous organic solvents.

• Ethanol precipitation: A prevalent method to desalt and concentrate DNA during experiments. With ethanol, monovalent cations are added to DNA to make the concentration of the solvent 70%. Ethanol disrupts DNA structure so that the molecule aggregates and precipitates out from the solution after centrifugation. Like the organic extraction process, it’s also a cost-effective technique, however time-consuming, to perform.
• Anion exchange chromatography method: This purification technique involves the binding of the negatively charged DNA phosphate backbone with the positively charged DEAE functionalized resins to precipitate DNA from the solution. This is achieved by using specific salt and pH conditions and stringent washing steps to remove contaminants like proteins and cellular debris. It provides high-quality DNA with high yield for workflows like DNA sequencing and transfection.
• Silica-column-based method: A conventional technique of DNA purification. It involves the use of chaotropic salts, which provide conditions in which the DNA binds to the silica column, and then elutes from the sample provided opposite condition — in low salt environments.
• Magnetic beads-based method: In this process DNA isolation and elution occur in a pH-dependent manner. At a low pH condition, the DNA binds to positively charged magnetic beads and at high pH, the beds release the molecule. It’s an effective technique for high automation of high-throughput processing.

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.

How To Perform DNA Purification?

Here’re the basic steps used in DNA purification:  

Cell lysate preparation

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:

• Enzymatic methods: Uses more structured starting materials to break tough cell walls. The most common enzymes used in the process include proteinase K, collagenase, lysozyme, zymolase, liticase, and lipase.
• Physical Methods: Includes crushing or grinding tough tissues. Some common methods include freezing and grinding samples under liquid nitrogen with a mortar and pestle. It’s generally used with plant cells.
• Chemical Methods: The most effortless technique to perform, commonly used in combination with other techniques. The chemicals used in the process include chaotropes (e.g., guanidine salts and alkaline solutions) and detergents (e.g., SDS).

Cleaning lysate

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.

DNA Isolation

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:

• Silica-Binding Chemistry
• Solution-Based Chemistry
• Ion Exchange Chemistry
• Cellulose-Binding Chemistry

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.

Washing

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.

Elution

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.

What Industries Use DNA Purification?

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.

Genetic Engineering

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.

Pharmaceutical

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.

Forensics

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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