To get the best results from the polymerase chain reaction (PCR), scientists rely on purification. Performing that process as well as possible depends on information and the right tools. Plus, scientists must think ahead.
“There are challenges to consider before and after purification of PCR products,” says Jordan Hunter, qPCR product manager at Analytik Jena. “Identifying the starting material and the appropriate purification method is very important.” For example, there are variations in endpoint PCR and real-time PCR, and some downstream processes can impact the results. “For example, endpoint PCR products obtained from TBE or TAE agarose gels can provide added specificity to collect the amplicons of interest, and allow for the removal of non-target specific PCR products before the purification process,” Hunter explains.
After selecting the source material, PCR purification can include methods involving columns, gels, ethanol precipitation, and so on. “Effective PCR purification removes all components that were used to facilitate amplification of the target sequence,” Hunter says. “The purification process then must remove enzymes, primers, short fragments, and salts from the reaction to provide pure product needed in downstream applications like next-generation sequencing.”
Image: The products from the polymerase chain should be as ready to use as possible, which often requires purification. Image courtesy of Analytik Jena
Some methods of PCR require more purification than others. As Hunter explains, “Methods that involve precipitation or washing with ethanol must be monitored closely to also prevent any residual amount of wash solutions used in the purification from inhibiting the downstream process.”
The purification of PCR products must also produce the desired molecules. So, Ryan Kemp, director of nucleic acids solutions at Zymo Research, notes that the process must result in “efficient recovery and the removal of unwanted fragment sizes.”
To get the desired results from PCR purification, scientists often rely on kits.
Considering kits
In shopping for a PCR-purification kit, many features should be considered. As Kemp explains, a kit should:
- effectively remove PCR inhibitors
- ensure maximum purity
- effectively enable selection of the desired fragment
- have effective recovery so that there is minimal loss of information in the next steps
- enable a very concentrated elution, which is ideal for things like library preparation.
As Hunter describes it, a kit should deliver “PCR cleanup products that provide the best rate of return for the fragment lengths required downstream,” he calls this “a priority that will ensure consistently obtainable product yields.” He adds that a product should work with a range of: “starting materials, material quantity, and elution volumes that can offer adjustment of product concentrations as needed.”
It’s worth taking the time to find the best kit. When Hunter talked more about the features that he recommends, he adds, “If these criteria are fulfilled, then a quick robust protocol that requires minimal steps and product manipulation is also ideal.”
Some kits even point out potential time savings. As an example, Cytiva describes its GFX PCR DNA and Gel Band Purification Kits as a “fast and easy-to-use method with less than 10 min hands-on time.”
What’s new?
Beyond the basics in kit features to consider, new methods allow more opportunities. “The most dramatic advance was the redesign of the spin-column to enable extremely low elution volume and no carryover,” says Kemp. For example, Zymo Research’s DNA Clean & Concentrator kits work with the Zymo-Spin column technology.
According to Hunter, “While the spin-column method of binding and elution of product has been generalized among kit protocols, advances in chemistry and automation have provided significant gains in nucleic-acid binding capacity, range of product fragment size, and the reduction of washing steps and buffer solutions needed,” he says. “Improvements in chemistry included in the innuPREP DOUBLEpure kit, for example, also allow for purification from different source materials including PCR reaction mixes and agarose gels in one kit.” He adds, “Ease of use, and the elimination of manual process steps where errors can occur are where we should see continuous improvement.”
Speeding up the steps
Some scientists seek ways to work without the need for purification. In some cases, that means looking for ways to simplify sample preparation for PCR.
A team of scientists in Japan, for example, wanted a faster way to study gene expression in potato tubers. They noted: “In the development of transgenic potato or of potato with other genome modifications … to improve tuber traits, analysis of the target gene is often difficult….” By using microtubers, the scientists reduced the growing by about 3–4 times, and they found that they could easily prepare RNA that could be used in PCR without purification.
During the COVID-19 pandemic, scientists and public-health experts seek increased speed in many processes, including PCR. Another research team in Japan looked for new speed in analytical methods applied to COVID. They wrote: “Rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for the diagnosis of coronavirus disease 2019 (COVID-19) and preventing the spread of the virus.”
This team of scientists tested the “2019 Novel Coronavirus Detection Kit” from Shimadzu. The team from Japan said that this kit “halves the detection time by eliminating the steps of RNA extraction and purification.” Plus, the researchers noted that this PCR-based test “effectively detects SARS-CoV-2 in all types of samples including saliva, while reducing the time required for detection, labor, and the risk of human error.”
In any PCR-based application, scientists need ways to get the best results. That can require purification all along the way or developing methods that eliminate that need. The better a PCR-based test works, though, the more reliable are the results. Likewise, the value of speed cannot be ignored, especially during a global public-health emergency like we’re experiencing now. So, the new tools and techniques described here all play fundamental roles in healthcare, as well as basic research.