High Throughput Nucleic Acid Purification Simplified

insights from industryBats Mutamba & Nans BodetField Support Automation ScientistsPromega Corporation

An interview with Bats Mutamba and Nans Bodet, conducted by James Ives

Please give an overview of the current gold standard in high throughput nucleic acid purification. What advantages and limitations are these for these methods?

Bats: The historical gold standard for nucleic acid isolation has been technology based off of a column workflow. In the regular workflow there is a lysis step, where the cells are taken and opened to expose the nucleic acid to the external environment, then a binding step onto a membrane on the column, followed by several wash steps to get rid of any cell debris, the proteins and other materials outside of the nucleic acids. In the final elution step, a buffer would be employed to dissociate the nucleic acid from the matrix of the column.

These workflows have since shifted to magnetic bead-based technologies, which essentially possess the same steps: lysis, binding, washing and elution, but this happens on a bead instead of on a column.

Column-based technologies have a few disadvantages: wells are prone to clogging with or without the use of vacuum manifolds. This can occur during the addition of buffers as well as numerous, cumbersome and time-consuming centrifugation steps to spin down the columns. This is not the case with the bead-based technologies, which are seen as much cleaner systems with shorter processing times.

Nans: Labs are demanding purity and flexibility for high throughput nucleic acid purification, while needing to complete difficult downstream analysis, for example with Next Gen Sequencing (“NGS”).

The magnetic based chemistry is the gold standard because it can provide a lot of purity, as you can select the appropriate type of magnetic resin to fit your application and fine tune for the most appropriate downstream applications. Magnetic beads are flexible in terms of scalability and sample input volume, as well as the numbers of samples that need to be run.

This is in comparison to other well-known techniques such as vacuum based separation, where the full plate needs to be used, diminishing the flexibility of the technique, and affecting sample volume. One advantage of the vacuum method is the speed, it can provide quick answers, but it doesn’t handle demanding downstream applications as well as magnetic bead-based chemistry.

How does the Maxwell® HT System work to purify DNA or RNA? What instrumentation does the Maxwell® HT System work with and at what scales?

Bats: The Maxwell® HT chemistry mentioned earlier is predominantly a bead-based technology to isolate nucleic acids, either DNA, RNA or total nucleic acid (TNA), which includes both DNA and RNA. If cells need to be lysed in the initial lysis step, specific buffers will be required, for example when isolating DNA from white blood cells.

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Then the nucleic acid is bound onto the magnetic beads (or resin, as we call it), which can have different types of surfaces. We tailor the binding chemistry to provide optimal capture of the molecules of interest. Once the nucleic acid binds to the magnetic beads there are three or four washes, which can be alcohol-based, but for some customers who have sensitive downstream assays, we have alternative buffers available.

The final step is the elution step, involving the detachment of the nucleic acid from the bead with an elution buffer, typically nuclease free water or a tris-EDTA buffer (TE buffer). If our customer’s downstream assays are sensitive to EDTA, which we know is a chelating agent, then we can use 25 mM Tris buffer.

The systems we have installed typically process from hundreds to thousands of samples. We are platform agnostic, which means that we integrate reagent chemistries onto any automated platform the customer desires. This gives us more flexibility without forcing our customers to decide on systems that may not be ideal for them. We offer recommendations and no hard requirements.

We have some Promega instrumentation which we use for demos or proof of concept studies, and we have worked with instrumentation companies from all the big players including Hamilton, Tecan, Beckman, ThermoFisher, Eppendorf and many others. There is a wide array of platforms that can be used for the chemistries and our team has diverse knowledge scripting or programming these platforms.

Nans: The beauty of the Maxwell® HT System is the well-proven chemistry, which can easily be demonstrated in the small Maxwell® RSC Instrument. We have plenty of experience with our system and we can scale the same chemistry to any kind of throughput.

Promega offers a customized solution to nucleic acid purification, please describe what you offer to your clients.

Nans: The customer or lab may have a very standard sample and we have the established solutions for it. However, most of the time, the samples being tested will be needed for a specific application, for example bacteria from feces, or the downstream assays might be very particular for this application, or the required volume is very different.

As we have a full grasp of the chemistry required, we can help the lab with a customized solution, by requesting that the lab sends us some of the samples for evaluation. We then work in our application labs in around the globe to test different chemistry solutions on the samples and define all the volumes required for the best performance of the downstream application.

Based on that, we are able to fine tune our final offer, we can provide a standard kit for this application with extra reagents if required or make a fully customized kit, with the same lot number for the whole year for the lab.

Bats: As Nans mentioned there is not any one customer that is going to want the exact same workflow with the exact same throughput, so Promega has two additional groups that are responsible for customization. Our FSS team (“Field Support Scientists”) works together with the Customs and OEM solution group as well as a Customs Order Department (COD), which handle bulk pricing, ordering and custom kit configurations.

For example, if you are a customer wanting to pursue a specific chemistry, and you do not want to go into your reagent bottles multiple times per run, we can package the reagents in a customized way for your lab. We work with our customers and our customer support teams to work on different kit configurations that are optimal for your workflow. If it is not a standard kit configuration, we have the ability to tailor-make reagents or packaging to help our customers, no matter the throughput level.

How does the Maxwell® HT Custom Nucleic Acid Purification Support Team save time and reduce stress?

Bats: The FSS team first works with the customer and local representative to develop an understanding of the requirements for laboratory workflows and automation needs. The team uses our expertise in automated systems and Promega chemistries to offer recommendations for the customer to consider and then will assist the customer in the lab to program the instrument, train the lab on using the instrument and chemistry and offer ongoing support.

Nans: We have a very well-established process, first in terms of the workflow documentation, we collect requirements then we have a statement of work where we define the expectations of the lab as we see and understand them. We share this with the lab before we go for the on-site implementation to double check that both expectations match. With this we save a lot of time.

We also save time thanks to our very large automation knowledge within the team, all the team colleagues have been working for a long time in the automation field, so we are able to support the lab in defining the best automation solution if they plan on purchasing an instrument or if they already have robots in their lab, by giving the best advice on which parameters should be used and which modules.

Thanks to our expertise we are also able to truly help the lab to implement the chemistry because we are not just selling one box of chemistry and sitting behind the customer to have them do their thing. We take control of the computer and define all the protocols manually without further help.

We are here to deliver biological performance, this is usually something that customers and the lab do not expect because of past bad experiences with other companies. However, in our case we are really here to deliver biological performance, and this helps decrease the stress level of the lab.

What kinds of HT purification problems has the team helped customers solve?

Bats: We often receive inquiries from laboratories who are either trying to re-task existing laboratory automation to a new process or are choosing to begin investing into laboratory automation.  Those customers may not have automation expertise and they appreciate the ability to consult with automation experts as they are considering various options.

As far as specific extraction problems, we are more than happy to help solve them. In our teams, we can always go on-site and work with a customer on their difficult sample types or the other alternative is the customer has the option to send samples to us where our Scientific Applications team will work on a solution for those particular sample types.

We will then send those samples back to the customer, and they can then test the isolates using their downstream assays. We pride ourselves as automation specialists in having the ability to help a customer on-site with scripting or programming.

Nans: In one case with a biotechnology company in France, they wanted to do plasmid purification, normally easy, but since their downstream process was to work on transfection and antibody proliferation, they needed a lot of plasmids—a really huge yield!

They needed to automate midipreps starting from 20-30 ml of cultures, something we hadn’t done before. However, since we have had lots of success in miniprep format in 96 wells, we tried that in our lab first with Promega cultures and samples. With the internally developed samples we tried to manually push the boundaries of our chemistry to see if it could handle the midiprep variation.

Once we had that working, we asked the customer if they could provide us with some of their culture to test differences between internal samples and external ones. We tested whether it worked manually with these samples and then, since we had some confidence in our chemistry and we have automation in our labs, we put the chemistry on the automated instrument in a 24 deep well format to handle the scale of volume similar to the midiprep.

After some weeks of development, we were able to develop a fine-tuned protocol to handle the midipreps and then went to the laboratory site in Marseille to implement and fine tune it further for the lab’s needs. This has been a very successful installation because the lab is very happy and we were able to write a combined poster together.

How is the team able to assist labs with any instrumentation platform?

Nans: Once you automate a chemistry on one platform it’s pretty straightforward to move that over to other platforms because the key issues are basically defining the proper liquid handling parameters, for example: Is this sample viscous or not? What should the incubation temperature and shaking speed be?

Once you have that and you have a basic knowledge of automation it’s easy to translate into any other automation language. Within the our team we all have previous industry experience. I worked for 10 years at Tecan before joining Promega and you will find similar experience in all of my colleagues on the FSS Team. Since the team was created 8 years ago, we have produced so many templates that we can use and then customize to any platform.

Bats: Our team is so diverse in terms of the combined knowledge we have gathered over the years on different platforms. The pipetting software may be very different in terms of programming but, by and large, if you have a good handle on the chemistry and if you can program on one of the bigger liquid handling platforms, then liquid handling as a whole is not as cumbersome as you might think.

As a group having that diverse background and knowledge in raw automation, we’re not going to dictate what platform a customer chooses for our chemistry. We do have recommendations for which automated platforms work better than others, some have more versatility or flexibility, but the take-home message here is that we are platform agnostic. We do not care which platform the customer uses with our chemistry.

What challenging sample types have the team encountered and been able to support?

Nans: Lately there was a focus on the microbiome. There’s a lot of interest in amplifying nucleic acids from feces, not only human feces but also mice and other species, which is typically difficult to handle because of the dirtiness of the sample, and it’s usually very difficult to gain a clean eluate.

We also have lots of requests for plant samples. Thanks to one of our specific chemistries, the Maxwell® RSC PureFood GMO and Authentication Kit, it’s easy to purify these difficult samples. We always have to carefully manage the sample storage, input and handling, but then we are quite successful in this field.

Bats: We have also had customers who have wanted to extract DNA from shell fish and various crustaceans, in the marine and biotech sector. We also have had requests to extract nucleic acid from fingernails and toenails! People often forget that there is a lot of DNA and RNA in the nails, as well as hair follicles. We have dealt with all of those types of samples.

The bottom line is even if we do not have experience with a specific sample type, the customers have the option of sending those samples through to us in Madison where we work hand in hand with scientists to get their extract to work.

What assurances are provided to customers about the quality of the Maxwell® HT Systems?

Nans: Promega as a company has a great track record in this field, so I would say this is the first level of assurance. Then, of course, we always like to double and triple check everything and we like to try the lab sample with our Maxwell® RSC system, which is very easy to handle and demonstrate on-site.

In either our or the client’s lab, we can test the sample in one hour allowing our customer to see for themselves if the chemistry is working as expected.

When a lab ships a sample to Madison or Lyon, where we can fine-tune the automation parameters of the experiment, we write a report and send the eluate back to the customer’s lab so they can also have a clear look at the DNA and RNA and see for themselves that it is working. If the lab is happy, we would then come on-site and repeat this work with their scientists and their platform, which gives a high amount of assurance.

Bats: We are able to demonstrate that our eluates are to the standard that the lab expects by testing the resulting sample. Our guarantee of success for our customers is that the chemistry does not lie.

For instance, with a nucleic acid purification chemistry, there are purity ratios for quality control of the samples. There are two ratios we look at when we isolate DNA and/or RNA, the first one is the A260-280 ratio which basically measures any protein contamination in your nucleic acid sample. When you test your samples, you can expect the ratio to be about 1.8 to 1.9. If there are proteins or other contamination in the sample, the ratio will be significantly lower i.e. 0.7 to 1.3. This tells you what kind of a sample you have and how successful the isolation went.

The second ratio is the A230-260 ratio, which essentially measures wash buffers or waste carried throughout the workflow. From the lysis step, binding step, washing step to the elution step, if any of those undesirable components are carried through the workflow that ratio will show it. We typically look for ratios of 1.7-2.1, and if it drops to 0.7-1.0 you know that there are high levels of buffer or waste materials carried through the workflow.

In addition to that we will then work with the customer on the downstream processes. Typically, if the system does work and the extractions were successful that should be demonstrated on those downstream assays.

What does the future hold for Promega and the Maxwell® HT team?

Nans: From a team perspective we have been quite successful, 5 years ago the team was just 2 people in the US and now we are 9 people and currently looking to hire a new person in Europe. We are expanding and we are also trying to educate people about automation throughout our organization to make them aware of the typical challenges of automation in the lab, so we can be even more successful in this field.

Bats: In terms of the future, we are excited about new technologies scientists are working on in academia, hospital and clinical labs, and pharma and biotech. For example, plasmid isolations for CRISPR, synthetic DNA production and blood banking, where huge amounts of blood are isolated to provide nucleic acid products to customers. We have seen lots of demand in these areas.

As Nans mentioned, our team is outstanding. I think as technology is running ahead, we are proactive and see where our chemistries and our automation can best be used.

Where can readers find more information?

https://www.promega.com/products/dna-purification-quantitation/high-throughput-nucleic-acid-purification/maxwell-ht-nucleic-acid-purification/

About Nans Bodet

Nans Bodet, is a Field Support Automation Scientist at Promega Corporation based in France and covering the EMEA for the last five years. He acquired a Master of Cellular Biology, physiology and Immunology at the Marseille Luminy University.

Prior to joining Promega, he worked at Tecan France as an Application Specialist and then joined Tecan Headquarters in Maennedorf (CH) to be Global Frontline Support and Key Account Manager. At Promega, Nans is focusing on combining Promega solutions with all kind of automation to ease laboratories challenges.

About Bats Mutamba

Bats Mutamba, a Field Support Automation Scientist with Promega Corporation for three years, was born and raised in Zimbabwe with a background in microbiology, biochemistry and biotechnology from Rhodes University, South Africa.

He moved to the US where he completed a Masters in Cellular and Molecular Nutrition from UNC Greensboro. At Duke University, he worked as a Research Analyst before being hired by Tecan as Product Specialist for Cell-Based Systems.

A while later, Bats went on to work for a nanotechnology company as a research scientist in product development, utilizing silver nanoparticles for various antimicrobial applications. He later worked for the CRO Pharmaceutical Product Development (PPD) in Richmond, VA as a senior scientist responsible for the development and validation of various assays using automated liquid handling.

He then relocated with his family to Phoenix, Arizona where he worked for Caris Life Sciences, a molecular diagnostics company specializing in cancer and personalized medicine. In his role as research scientist he was purposed with developing and automating laboratory developed tests (LDTs) for the early detection and diagnosis of breast and prostate cancer.

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