How do you purify nucleic acids?

How do you purify nucleic acids?

Scientist pipetting a sample into the well of a gel tray to perform agarose gel electrophoresis

Are you in the process of setting up a new lab and wondering what you need for nucleic acid purification? Or have you found that your samples just aren't pure enough for your downstream application? Then you've come to the right place. This article represents the fifth and last part of our series 'From samples to pure nucleic acids'. After covering the topics of sample collectionnucleic acid extraction, the set-up of a PCR lab, and nucleic acid quantification and visualization, we now turn to the different purification methods. Besides the advantages and disadvantages of each technique, we'll also explain what equipment you need.

Table of contents
Illustration explaining that you need to extract NA, set up a PCR, quantify NA and purify them to get from samples to pure nucleic acids

In contrast to nucleic acid extraction, nucleic acid purification doesn't include any lysis steps to break the cell membrane and liberate nucleic acids. Instead, it involves the clean-up of your samples, e.g. to effectively remove all components that were used to facilitate amplification of the target sequence during PCR.

1    Ethanol and isopropanol precipitation

The first purification method we'll have a closer look at is ethanol and isopropanol precipitation. This is the method of choice to purify genomic DNA, and can be used to concentrate and de-salt nucleic acid samples after applications such as CsCl density gradient centrifugations with EthBr, phenol-chloroform extractions, digestions, and PCRs.

1.1    How does it work?

Ethanol and isopropanol precipitation are all about solubility. Water and nucleic acids are both polar molecules, which is why nucleic acids dissolve easily in water. To precipitate them, you can either use ethanol or isopropanol.

For ethanol precipitation, you need to add twice the sample volume of ice-cold 96 % ethanol, and salt (commonly sodium acetate) to the solution. Ethanol lowers the dielectric constant, allowing the negative charges on the sugar-phosphate backbone to be neutralized by the Na+ ions of sodium acetate. Since the nucleic acids are now less hydrophilic, they will drop out of the solution when you incubate the mixture on ice. Then centrifuge your sample to separate the nucleic acids from the rest, and wash the pellet in cold 70 % ethanol to remove any residual salt. Centrifuge the sample a second time, remove the ethanol, allow the nucleic acid pellet to dry, and resuspend it in a clean aqueous buffer.1 To dry the nucleic acid pellet, you can either place the tube (with the lid open) in a laminar flow hood for several hours, or use a vacuum centrifuge. The method you choose is up to you, but you have to ensure that the pellet is completely dry to avoid residual ethanol negatively affecting your downstream applications.

Isopropanol precipitation is very similar. The only differences are that you can skip the incubation on ice step, and replace ice-cold ethanol with room-temperature isopropanol for the first step. Regarding the volume of isopropanol, an amount equal to the sample volume is sufficient.2

Whether ethanol or isopropanol is more suitable depends on your sample volume, concentration, and the size of your nucleic acid fragments. If you have a large sample volume, it may be impossible to add twice the sample volume of ethanol into the tube. Isopropanol precipitation is also preferable for the precipitation of larger nucleic acid fragments and lower sample concentrations, as nucleic acids are less soluble in isopropanol. On top of that, isopropanol precipitation is the faster method as you don't need to incubate your samples before centrifugation.2

1.2    Advantages and disadvantages

Ethanol and isopropanol precipitation aren't costly at all, as ethanol, isopropanol and sodium acetate are quite cheap, and the process provides a good yield of high purity nucleic acids. It is, however, a very time-consuming process and, as it needs to be performed manually, highly variable. Consequently, low reproducibility can be a problem.

1.3    Equipment needed

All you need for ethanol or isopropanol precipitation is a centrifuge and a laminar flow hood, or a vacuum centrifuge to dry the pellet.

2    Gel electrophoresis and spin column purification

As gel electrophoresis separates the nucleic acids based on their length, you can use this method to separate nucleic acids of interest from other nucleic acid types and contaminants.

2.1    How does it work?

First of all, you need to run a gel. More detailed information on how to perform agarose gel electrophoresis can be found in section 1 of our article on nucleic acid quantification and visualization.

Agarose gel with clearly visible nucleic acid bands

After visualizing your gel, use a sharp scalpel to excise the nucleic acid band of interest. Always remember to wear appropriate PPE (face shield and gloves) for this step, especially when using UV light for the visualization of your bands. Then, purify your nucleic acid bands from the TAE or TBE-buffered agarose gel by using a suitable spin column purification kit. As the various kits available slightly differ from one another, you should carefully follow the manufacturer's instructions. Usually, you need to weigh the nucleic acid bands, add a specified amount of buffer for every 100 mg of gel slice, and heat the mixture to solubilize the agarose.3,4 You then transfer your samples to the spin columns and purify the nucleic acids using binding, washing and elution steps. More detailed information on how spin column protocols work can be found in section 2.2.2.1 of our article on nucleic acid extraction.

2.2    Advantages and disadvantages

The huge advantage of this nucleic acid purification method is that agarose doesn't denature the nucleic acids, making it easy to recover them from the gel without any harm. It is, however, not suitable for high throughput labs, because running a gel is very time consuming, and is limited to a low number of samples.

2.3    Equipment needed

Compared to other purification methods, you need a lot of different instruments for this workflow. Agarose gel electrophoresis requires a gel electrophoresis system, an external power supply and a biosafety cabinet, as you'll be working with hazardous intercalating dyes. Spin column purification is less demanding, as you only need a centrifuge.

3    Spin column purification

Spin columns are not only used to extract nucleic acids from lysed samples, but also to purify them, e.g. after a PCR reaction to remove salts, enzymes, primers, primer dimers and nucleotides that may inhibit subsequent applications.

3.1    How does it work?

As explained above, spin column purification protocols consist of binding, washing and elution steps. After transferring your samples into the spin columns, you centrifuge them to bind the nucleic acids to the membrane inside the column. This allows unwanted components to pass through. Several additional centrifugation steps with a wash buffer remove residual unwanted materials, and a final centrifugation step with an elution buffer liberates the nucleic acids from the membrane.

3.2    Advantages and disadvantages

As you can see, this purification method is quick and easy. On top of that, it can be adapted to your sample number, as you could also use 96 well silica membrane plates instead of single spin columns. However, the membrane may get clogged and, as a minimum elution volume of 30-50 μl is required, you may get rather low nucleic acid concentrations.

3.3    Equipment needed

The only piece of equipment needed for spin column purification is a centrifuge, unless you work in the 96 well format and prefer to use a vacuum manifold with a pump, which is also possible.

4    Magnetic bead purification

Just like spin columns, magnetic beads can be used either for the extraction of nucleic acids from lysed samples or for their purification. For example, you can use magnetic beads to remove salts, enzymes, primers, primer dimers and nucleotides from PCR products that would otherwise inhibit subsequent applications.

4.1    How does it work?

The purification workflow with magnetic beads is very similar to the extraction workflow. You first add magnetic beads that bind the nucleic acids to your samples.

AMPure XP by Beckman Coulter for PCR Purification

You then place the tubes on a magnet, remove the supernatant containing unwanted, unbound material. You repeat this step several times, replacing the wash buffer in between. In the end, you add an elution buffer and transfer the samples to a different vessel.

In contrast to extraction protocols with magnetic beads, only nucleic acid fragments of a particular length bind to the beads during purification. This size exclusion mechanism is achieved by creating the perfect binding conditions for the nucleic acid fragments of interest, by varying the buffers, salts, and their concentrations, and therefore the hydrophilicity/hydrophobicity.

4.2    Advantages and disadvantages

A huge advantage of magnetic bead purification is that the equipment needed can be chosen based on your budget.

Another convenience of magnetic bead purification is that you can work with lower elution volumes than for spin column purification, and it is more easily scalable, as you can use it with 384 well plates.

4.3    Equipment needed

As described, you can match the equipment for magnetic bead purification to your budget. Either buy a magnetic stand if you have a limited budget, get a benchtop pipetting robot or a 96 or 384 channel pipette if you have more money available, or go with a dedicated purification system.

5    Sephadex® purification

Sephadex purification can be used to purify nucleic acids from smaller molecules, e.g. primers, nucleotides or dyes.

5.1    How does it work?

Sephadex is a gel filtration resin made of dextran crosslinked by epichlorohydrin.5 To prepare the resin, you need to add Sephadex powder to spin columns, rehydrate it with water, and centrifuge the columns to eliminate excess water. Before spinning the columns a second time, add your samples on top of the resin. During centrifugation, larger molecules will easily pass the resin and elute, whereas smaller molecules will get trapped in the pores of the dextran beads. The size exclusion capability of the beads depends on the Sephadex type you choose. For example, Sephadex G-25 Medium5 can be used to purify nucleic acids with a molecular weight of >5000, and G-50 Medium6 is suitable for molecules with a molecular weight of >30000.

To speed up this workflow, opt for spin columns prepacked with Sephadex instead of creating them yourself, or work in a 96 well filter plate.

5.2    Advantages and disadvantages

Just like spin column purification, this method can be adapted for your throughput needs, by either performing it in spin columns or in 96 well filter plates. The absence of a molecule-matrix binding step also prevents unnecessary damage to the nucleic acids,7 making it a rather gentle purification method. However, it's quite time consuming, especially when preparing your spin columns or filter plates yourself – Sephadex has to rehydrate for about three hours – and can't be automated.

5.3    Equipment needed

The only piece of equipment needed for Sephadex purification is a centrifuge.

6    Enzymatic approaches

Some manufacturers offer enzymatic approaches to clean up your PCR products if your subsequent application – e.g. Sanger sequencing, next generation sequencing or SNP analysis – requires your samples to be free from unincorporated primers and dNTPs.

6.1    How does it work?

Enzymatic approaches consist of only two steps. First, you add an enzyme mix to your samples and incubate them for 15 minutes at 37 °C. During incubation, the first enzyme in the mix, exonuclease I, will digest excess primers, and the second enzyme, alkaline phosphatase, will dephosphorylate dNTPs that were not consumed during PCR. Once the enzymes have served their purpose, you can heat up your samples to 80 °C for another 15 minutes to deactivate the enzymes.8,9,10

6.2    Advantages and disadvantages

In addition to being quick and easy, this method results in no sample loss, and can be easily adapted to your sample number. Its only disadvantage is that it can only be used to clean up PCR products from primers and dNTPs, and does not remove any other contaminants.

6.3    Equipment needed

All you need for enzymatic PCR cleanups is an incubator.

7    Conclusion

As you can see, there is a nucleic acid purification method available for every sample type, downstream application and budget. We hope that this article helped you determine which one to choose for your specific needs.

And if you haven't only come here for tips on nucleic acid purification, you might also be interested in learning about what you need to collect samplesextract nucleic acids, perform PCR reactions and quantify nucleic acids. We put together this five-part series when we were in the process of equipping a new in-house lab that now looks like this:

Laboratory how it looked before and how it looked after we equipped it with several instruments

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