Even “Boring” Ideas are Surprisingly Deep

It’s easy, in biology, to become enamored by the frontier. But the old, boring stuff often hides many layers of complexity.

Photo by me.

I.

Many ideas that seem “simple” on their surface are surprisingly deep. I suspect this is true in every scientific field, and it’s certainly true in biology.

Polymerase chain reaction, or PCR, is one example. PCR wasn’t mundane when scientists invented it in the 1970s, but even high schoolers learn it today. The technique seems simple, in part, because teachers present old methods matter-of-factly.

Maybe you learned about it like this: heat a DNA sample to around 95°C to “melt” the two strands apart. When the temperature cools, small DNA snippets called primers attach to the freed strands. Next, heat the sample again — this time to about 70°C — so an enzyme called DNA polymerase can latch onto each DNA strand and copy it. When it’s done, heat the sample back to 95°C and restart the cycle. The key thing to remember is that heat does the work of separating the strands, and the rest of PCR follows.

In a recent essay, I made the grandiose claim that PCR is a “near-optimal” technology. My core argument was that DNA polymerases are already fast, so any further speed improvements would come from the temperature cycling steps. If we could instantaneously change the temperature of DNA samples (using lasers, for example), we could shave about 20 percent off the time needed to do PCR. This is only a modest improvement, though; hence my claim that PCR is “near-optimal.”¹

OH BOY WAS I WRONG!

After the essay went out, Momčilo Gavrilov — a biophysicist at Johns Hopkins University and co-founder of SHARP Diagnostics — reached out and told me he’d invented a way to do PCR at a single, fixed temperature. Instead of using heat to break apart the two DNA strands, Gavrilov uses a helicase enzyme that physically separates them, letting primers bind and polymerase copy the DNA. You just mix the ingredients and incubate them at a single temperature between 37°C and 65°C.

Gavrilov’s method seems to be a major improvement over standard PCR for three reasons. First, it works in existing thermocyclers, or in tiny devices that you could carry in a pocket. Second, isothermal PCR can be much faster; without the temperature shifts, amplification runs continuously, with no pauses between steps. And finally (this is the most important thing), ditching the high temperatures means you can drastically expand the amount of “chemistry” you can do during PCR. (For example, by ditching the high heat, biologists can now use a wider palette of polymerases for PCR. They are no longer limited to only heat-stable options.)

In short, I no longer believe that PCR was, in fact, a near-optimal technology. Even methods that seem “solved” or simple can, upon deeper research, turn out to be surprisingly deep.

II.

Isothermal PCR isn’t a new idea.

In his 1987 PCR patent, Kary Mullis suggested using an enzyme to mechanically separate DNA strands, instead of heat.² He speculated that helicase, a motor protein that unwinds DNA during genome replication, might work. At the time, though, nobody had yet discovered a helicase that could pull apart DNA strands with blunt ends (where both strands “line up” at the same point); the enzymes needed a “loose” strand of DNA to grip onto.

In 2004, New England Biolabs began shipping kits for helicase-dependent amplification, or HDA, an isothermal method built around Mullis’s original idea. The problem with HDA, though, is that the helicase was too weak. It would open up DNA, unwind the first 150 bases of DNA, and then fall off. The polymerase enzyme would begin copying this “opened” DNA, but couldn’t proceed any further than the helicase. These limits meant that HDA could only amplify small snippets of DNA. BioHelix, a spinout from New England Biolabs, commercialized the method and used it to make two FDA-approved diagnostics, including for herpes simplex virus.³

The “weak helicase” problem was later solved by scientists at Johns Hopkins. In 2015, Taekjip Ha’s lab described a “superhelicase” they had engineered. The enzyme was locked into its “unwinding” configuration, such that it couldn’t slip off the DNA. This superhelicase could open up DNA strands stretching 6,000 bases or more, but required a single-stranded DNA overhang to initiate that unwinding. It didn’t work on blunt end DNA!

A few years later, in 2022, Gavrilov engineered PcrA M6, a helicase from a “heat-loving” microbe that can open up blunt-ended DNA. This helicase — like the original superhelicase — was locked into its unwinding conformation, such that it could be used on long stretches of DNA. The helicase opens about 150 base pairs of DNA per second, but it’s likely possible to engineer a helicase that moves much faster.

Now, putting everything together, the isothermal PCR works like this:

First, mix together the DNA sequence (to be amplified) with the PcrA M6 helicase, Bst polymerase, primers, nucleotides, and SSB. Bst is a polymerase from a bacterium called Bacillus stearothermophilus. It works at moderate temperatures and, importantly, can separate paired DNA strands as it copies them, a property called “strand-displacement.” SSB, or single-strand DNA-binding protein, is a small protein that grabs onto single strands of DNA and keeps them from re-pairing. SSB is also what allows PcrA M6 to grab onto blunt-ended DNA in the first place.

Once everything is mixed together, the reaction starts. The helicase pries open a short stretch of the DNA, SSB coats the freed strands to keep them apart, primers diffuse in and bind to their target sites, and the polymerase starts copying.

But the polymerase, Bst, moves much faster than the helicase, PcrA M6. When the polymerase catches up to the helicase, it knocks it off the DNA and keeps going on its own, separating paired bases as it copies them. The displaced helicase is then free to bind to a fresh copy of DNA. This cycle repeats continuously.⁴

Gavrilov says his technique can amplify DNA fragments up to 6,000 bases long, or can double the amount of a 200-base fragment of DNA in under a minute. The reaction works anywhere from 30 to 65°C with standard PCR primers. In other words, it is a drop-in replacement for normal PCR.

Ahis is important! In my prior essay, I argued that many biologists do not adopt newer methods (like photonic PCR) because the switching cost is too high. Buying new machines and troubleshooting protocols is a lot of work, and the payoff is usually minor. Why bother, then, if an existing setup already works? Gavrilov’s method, though, can run the same type of reaction in the same thermocyclers that scientists already have, using the same primers. The switching cost is low, which makes adoption much easier.

III.

A “true” isothermal PCR method now exists, one that works on long DNA sequences. What can we do with it?

First, we can run PCR with much better polymerases. Biologists have historically used Taq (or engineered heat-stable polymerases, like Phusion) mostly because they survive at 95°C. But these are not the best or fastest polymerases available! The 95°C step basically acts as an artificial filter that limits PCR only to heat-tolerant polymerases, rather than allowing biologists to work with the millions of polymerases that only work at lower temperatures. The native E. coliDNA polymerase copies about 1,000 nucleotides per second and is highly accurate. We can presumably find polymerases that move faster still, and engineer them to do strand-displacement if needed. At high speeds, it should be possible to make billions of copies of even a long DNA sequence (3,000+ bases) in just a few minutes.

Second, we can expand the “chemical space” of PCR. Without the 95°C step, it’s trivial to add enzymes to the tube that would normally be destroyed by high heat.

Consider DNA methyltransferase, an enzyme that tags cytosines in DNA with methyl groups, which determines which genes get switched on or off. Methyltransferases are destroyed at high temperatures, which means that PCR can copy methylated DNA but doesn’t preserve that methylation as the DNA is copied. The amplified product comes out “naked,” in other words, without methyl groups. With isothermal PCR, you can include methyltransferase in the reaction mix and, as each new strand is copied, the enzyme writes the same methylation pattern onto the newly-copied strands.

Third, many DNA sequences can’t easily be amplified with normal PCR, because highly repetitive regions (like the CAG repeats in Huntington’s) form hairpins during cooling. In a 2024 preprint, however, researchers showed that this isothermal method could amplify a 561 base pair sequence containing 91 percent As and Ts, and even amplify stretches of DNA that contain 200 consecutive AT base pairs. Normal PCR couldn’t amplify any of it.

Finally — the thing I’m most excited about — is that isothermal PCR is just more portable. By getting rid of the normal heating and cooling of PCR, you no longer need thermocyclers, with their big metal blocks and fans and electrical cords. Instead, you can run isothermal PCR using a small heater, roughly the size of a coin, that holds a tube at 65°C for many hours using minimal electricity.

The reagents are also more portable, too. Without the heat-stable polymerases, researchers can instead choose enzymes that freeze-dry easily for long-term storage at room temperature. The helicase, SSB, primers, and dNTPs can all be stored as powders, according to Gavrilov. ATP, the least stable ingredient, can also be stored as a powder. The whole reaction can therefore ship in a small tube, at room temperature, and then be rehydrated with a drop of water. It’s easy to imagine a portable and cheap diagnostic tool that could detect dozens of different pathogens — in soil or human blood, say — in just a few minutes.⁵

My prior essay made arguments about PCR based on its historical “shape.” I considered all the steps involved and then reasoned through ways to make each one faster or cheaper. This is a natural way of thinking, but it tends to yield only incremental improvements. It doesn’t easily reveal entirely new ways of thinking, or out-of-the-box solutions like “what if we didn’t change the temperature at all?”

It’s easy, in biology, to become enamored by the frontier. PCR is forty years old and taught to high schoolers, so why bother writing about it? Surely there’s no room left to improve. But the old, boring stuff often hides many layers of complexity, and if we haven’t yet optimized a forty-year-old technique, what makes us think we’re anywhere close to understanding the frontier? Simple ideas are rarely simple.

1. Some readers told me that my claims about polymerase speeds were too low. But the published data on polymerase speeds is really bad! Some companies advertise rates as high as 2–5kb per second, which truly boggles my mind. I’ve never seen actual data to support such claims, and think biology ought to produce real benchmarks for basic claims like this.

2. It’s mentioned at least three times in the patent. One quote says, “…the reaction mixture may contain, in addition to the nucleic acid strand(s) containing the desired sequence, the strand-separating enzyme (e.g., helicase), an appropriate energy source for the strand-separating enzyme, such as rATP, the four nucleotides, the oligonucleotide primers in molar excess, and the inducing agent...” This is an identical match to what Gavrilov made nearly 40 years later.

3. There are other isothermal ways to amplify DNA. LAMP, invented in 2000, skips strand separation entirely. It uses a polymerase that moves through paired bases as it copies, but requires four to six primers designed so that the new DNA folds back on itself to create hairpin loops. Each loop creates a fresh site for the next primer, and the reaction cascades. The downside is that it’s tedious to design these primers, and the end result is these tangled stem-loops that you can detect (such as for diagnostics) but can’t easily clone or sequence. RPA, invented in 2006, uses a recombinase, an enzyme bacteria use for DNA repair, to slip primers directly into the double helix without pulling the strands apart. RPA runs at body temperature, but it needs unusually long primers (30 to 38 bases) that tend to fold up on themselves, and like HDA, it caps out around 100 to 200 base pairs. Neither of these methods, therefore, are a direct replacement for PCR.

4. It’s important for the polymerase to be strand-displacing because it moves much faster than the helicase. Remember that this engineered “superhelicase” only moves at 120–150 bases per second, whereas a good polymerase can move at 1,000+ bases in the same amount of time. Some polymerases are not strand-displacing, though, and so their speeds would be bottlenecked by the helicase.

5. The real bottleneck is probably economics. Even a mundane diagnostic, like for lead poisoning, is way too expensive to use and there is little incentive for companies to make better options. The returns on diagnostics are not nearly as good as those for a new cancer drug, for example, and so many companies don’t work on these problems.