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Penn State Researchers Offering License-Free DNA Ladders for Electrophoresis Studies

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NEW YORK (GenomeWeb) – An undergraduate research team at Pennsylvania State University has developed an inexpensive method to produce reference molecular weight markers, or DNA ladders, for gel electrophoresis studies.

The method, published late last week in the journal Nature Scientific Reports, uses two plasmids developed by the Penn State team which have the ability to produce DNA fragments ranging from 50 to 10,000 base pairs in length.

In addition, the researchers are making the plasmids available license-free to non-profit academic users, which they hope will provide users with a cheaper alternative to current commercial options.

"The original way that people made these standards twenty or so years ago was taking something like bacteriophage DNA and cutting it up," said Song Tan, professor of biochemistry and molecular biology at Penn State, the corresponding author and leader of the research group. "You knew how long the fragments were. They weren't round numbers but that was fine. Over the last ten or twenty years, companies have been selling their DNA ladders, which were nice round numbers. A lot of people are really comfortable using them, but they are quite expensive."

The Penn State team's plasmids offer a way for researchers to get DNA ladders with round numbers — those with exact measurements such as 100 or 1,000 base pairs — at a fraction of the cost of commercial options. The method would cost between $5 and $30, depending upon exact reagents and prep used, to produce 1,000 lanes worth of DNA ladders. Tan explained that with homemade plasmid prep the cost ranges between $5 and $7, but with a commercial plasmid prep kit, the cost jumps up to between $20 and $30. 

This is significantly cheaper than commercial options, which cost between $200 and $500, according to the researchers. The paper specifically mentions New England Biolabs' 1-kb ladder, 100-bp ladder, and 2-Log ladder products which are priced at $212, $488, and $244, respectively for 1,000 lanes of ladders. The researchers also mentioned Thermo Fisher Scientific's GeneRuler 1-kb ladder and 100-bp ladder which are priced at $282 and $466, respectively, for 1,000 lanes worth of ladders.

To produce the plasmids for the DNA ladders, the Penn State team first selected the pUC9 cloning vector as the backbone of the molecular marker because it is a high copy number plasmid, and is free of licensing restrictions to the best of the researchers' knowledge. In order to create a wide range of round numbers, the team separated the DNA ladder into two separate plasmids, pPSU1 and pPSU2. The researchers created both plasmids through a series of cloning steps, then generated the molecular weight fragments through complete restriction digestion.

The pPSU1 plasmid contains a number of EcoRV fragments ranging from 500 to 5,000 base pairs in length, as well as PstI fragments pairs ranging from 500 to 4,100 base pairs in length within 10,000 total base pairs. The pPSU2 plasmid contains the remaining EcoRV 1-kilobase ladder fragments, and the PstI 100-base pair ladder fragments ranging from 50 to 4,100 base pairs within 7,750 total base pairs.

The study describes exact procedures for how to process the plasmids, which Tan and his colleagues have made available through AddGene, a nonprofit plasmid repository. They are also in the process of depositing the plasmids in a different plasmid repository called DNASU, which is maintained by the Arizona State University Biodesign Institute and which Tan said is used by the National Institutes of Health.

The researchers also noted that they are specifically making the plasmids available to the nonprofit academic community without licensing requirements. "We have no plans/intentions whatsoever of making this a commercial product," Tan added. "That goes against our goal here, to make this a reagent available to the community."

Madhuri Hegde, VP and CSO of PerkinElmer's Global Lab Services who was not associated with the study, said that the method to make DNA ladders, something she uses all the time in her lab, "seems to be pretty robust" and that it is a "solid paper."

"What they have missed in [their calculation] is the labor, the overhead, and the cost to control quality," said Hegde, who also serves as an adjunct professor at Emory University's School of Medicine. She notes that it's not clear whether the final cost of in-house production is actually cheaper than commercial options once those factors are taken into consideration. "Ladders are not a big expense in the lab," Hegde added. "I've ordered [them] twice a year in my academic lab, and once every quarter in my clinical lab."

She also noted that it won't be clear how reproducible the results are unless someone tries to reproduce the ladders independent of the Penn State researchers.

Tan recognized that the Penn State plasmids might not be an option that every lab would want to pursue, since in the grand scheme of things the commercial DNA ladders aren't prohibitively expensive. "What we are saying is we want to offer [the community] an alternative, an option that if you would like to have high-quality ladders, but you don't want to have to buy a commercial one, here is way that you can make it for yourself at about a fiftieth of the cost," Tan explained.

The researchers feel that this might give small labs and labs with more limited resources an opportunity to have these tools at their disposal. Tan also adds that the creation of these plasmids could be used as a teaching tool in college labs, and possibly even high school labs.

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