NEW YORK – A team of researchers at Johns Hopkins University has developed a new PCR method that can function at a single temperature. The team hopes to launch a startup to commercialize home-use molecular diagnostics based on the method.
Helicases are molecular motors that can unzip nucleic acids, holding strands apart to enable DNA repair or replication. They are typically stably engaged with DNA for about five base pairs. But, in 2015 biophysicist Taekjip Ha and his team at JHU used intramolecular cross-linking to engineer a helicase that could unwind thousands of base pairs without falling off.
"We made a super enzyme that doesn't exist in nature, and we began to think about potential biotechnological applications," Ha said in an interview.
Ha and colleagues then took the method further, combining it with a single-stranded DNA binding protein, or SSB, to prevent reannealing. The results were reported in a study published last week in Nature Communications.
With SSB-Helicase Assisted Rapid PCR, or SHARP, "you can use the super helicase to unwind the DNA to load the next round of primers, instead of using heating in a PCR machine to denature DNA," Ha said. And, the next cycle can continue even before the previous cycle has been completed.
The reaction is isothermal, with temperature held at a constant 65° C. This temperature is just a bit hotter than extremely hot tap water, making the method readily useable in many types of settings.
SHARP also differs from other isothermal methods. Loop-mediated isothermal amplification "makes a Christmas tree" end product rather than useful DNA, Ha said, while recombinase polymerase amplification can yield DNA, but only short stretches of about 200 base pairs. Another isothermal method, called helicase dependent amplification, uses helicases and has been deployed commercially and academically in diagnostics development, but Ha noted that it only amplifies stretches of about 100 base pairs.
The SHARP enzyme can generate DNA molecules as long as 6,000 base pairs, Ha said, and it is easy to make since it only needs to be expressed in Escherichia coli.
The SHARP method can be used with many types of PCR reactions, but may need tweaking of some reagents. In particular, "we tested several DNA polymerases, and we found one that works well with our assay," Ha said.
In the Nat Comm study, the team showed the method could produce functional DNA in the form of a plasmid that confers antibiotic resistance to cells. They also used it to amplify fragments of genomic DNA.
And, "we demonstrated that, after you do Cas9-based gene editing, we can amplify the relevant genomic area using our method and run Sanger sequencing or next-generation sequencing to read the outcome of the editing," Ha said.
All of the reagents that are used in the method, except for the super helicase, are not currently protected by IP and are easy to make in large volumes, Ha said, so that the most expensive part of the method might be the investment in a hot water bath. Overall, it will likely be a very low-cost technology, he added.
Momcilo Gavrilov, a researcher in the Ha lab and R&D scientist at Honeywell developing diagnostics technologies, is working on a potential commercialization plan for the SHARP technology along with Ha lab graduate student Taylor Cottle.
In an email, Gavrilov said Johns Hopkins' Technology Ventures office currently has the capability to license the technology, and, "we are working closely with JHTV to find out more about the conditions under which we can license the technology for a potential startup."
The startup will be called SHARP Diagnostics, and the intention is to commercialize the method in increments, first with a SHARP kit for research labs then expanding into home diagnostic testing utilizing the method. The team recently won an award for their business plan at the JHU's HopStart pitch competition, Gavrilov said.
Ha noted that his group has also seen a remarkable amount of interest in the method since the Nat Comm paper came out last week.
The study has been downloaded nearly 40,000 times, and a tweet linking to the SHARP study — with the provocative text, "The end of PCR machines?" — has been retweeted more than 2,000 times.
"We have also received several requests for the materials," Ha said. He and his team are already providing the super helicase to others who want to test it, sending out aliquots or plasmids to labs and companies, and Ha has also been contacted by a venture capital firm that spotted the study and is considering investing.
"For a single paper, it generated quite a bit of interest in a short period," Ha noted.
That said, "if you can replace PCR machines with this method in most things that people use PCR for, this could be big," he added.