RIP Kary Mullis, Father of PCR

When the name Kary Mullis popped up in my news feed on Monday, I was excited to read what I thought would be an update on the renegade inventor I’d met years ago at a small biotech gathering in San Diego. Back then, in the late 1980s, I’d interviewed him for Genetic Engineering News, where I had the gene amplification beat – a field that began with the polymerase chain reaction, aka PCR.

An Eclectic Technology

Kary Mullis died on Monday, August 12, of heart disease and respiratory failure. He was so quirky that obituaries, like the one in the LA Times, led off with such descriptors as “LSD-dropping, climate-change-denying, astrology-believing, board surfing.” That obit calls PCR a “discovery.” But the technology wasn’t laying around waiting for someone to find it, like an ancient skull. Instead, it was an invention deduced from the scrutinizing the mechanics of DNA replication.

Other obits repeat the mantra that Mullis was an “untamed genius,” a phrase oddly the opposite of an oft-echoed presidential boast.

I bought into the Mullis mythology, relating the PCR origin tale in the dozen editions of my human genetics textbook:

“PCR was born in the mind of Kary Mullis on a moonlit night in northern California in 1983. As he drove through the hills, Mullis was thinking about the precision of DNA replication, and a way to tap into it popped into his mind. He excitedly explained his idea to his girlfriend and then went home to think it through. “It was difficult for me to sleep with deoxyribonuclear bombs exploding in my brain,” he wrote much later. (A quote so common that I can’t find the original source, which I think was Scientific American.)

The idea behind PCR was so simple that Mullis had trouble convincing his superiors at Cetus Corporation that he was onto something. Over the next year, he used the technique to amplify a well-studied gene. Mullis published that landmark paper in 1985 (on amplifying the sickle cell mutation) and filed patent applications, launching the field of DNA amplification. He received a $10,000 bonus for his invention, which the company sold to another company for $300 million. Mullis did, however, win a Nobel Prize in 1993.”

Some biotech friends tell me that Mullis’s story didn’t unfold quite so neatly, dramatically, and independently. And variations on the theme were well underway, according to this article I wrote for The Scientist in 1991. Still, PCR’s importance and ubiquity are clear – anyone who’s had a rapid strep or flu test has benefited from it.

Back in the 1980s, Mullis probably didn’t foresee PCR identifying the victims of the 9/11 terrorist attacks and other atrocities and natural disasters. The impact on forensics has arguably been as profound as the effect on diagnostics.

The invention of PCR came just after Sir Alec Jeffreys introduced the first DNA fingerprinting (now called profiling) technology. PCR extended DNA profiling to vanishingly small forensic specimens. The technology became entrenched, a buzzword by the time Olivia Benson and company tossed the acronym around on Law and Order: SVU. 

With each edition of my textbook, I curate the list of applications, so here’s some of PCR’s greatest hits. It has been used on:

• A preserved quagga (a relative of the zebra) and a marsupial wolf, both extinct.
• Poached moose meat in hamburger.
• A cremated man, from skin cells left in his electric shaver, to diagnose an inherited disease in his children.
• The brain of a 7,000-year-old human mummy.
• The digestive tracts of carnivores, to reveal food web interactions.
• Roadkills and carcasses washed ashore, to identify locally threatened species.
• Products illegally made from endangered species.
• Genetically modified bacteria released in field tests, to follow their dispersion.
• One cell of an 8-celled human embryo to detect a disease-causing mutation.
• Remains in Jesse James’s grave, to make a positive identification.
• The intestines of genital crab lice on a rape victim, which matched the DNA of the suspect.
• Fur from Snowball, a cat that linked a murder suspect to a crime.

How PCR Works

To picture PCR, imagine a row of aligned couples at a dance where lots of other folks are standing around, unpaired, a little like the gym scene in West Side Story. People keep arriving. The couples part and draw in new partners, over and over, until the gym fills with twirling pairs. Each new dancer wears a distinctive item, like a red scarf, to be noticeable. Not a perfect analogy to PCR DNA amplification, but close.

The “P” in PCR stands for “polymerase,” the enzyme that replicates DNA by adding the new dance partners. (“ase” indicates an enzyme; DNA is a polymer, a molecule of repeating units). DNA polymerase – DNAP – guides the doubling of DNA each time a cell divides, bringing in new DNA bases to form the “daughter” helices.

Mullis invented the basics of PCR in 1983, and the patent issued in 1986 (which now eerily reads “2019-08-12 Application status is Expired – Lifetime). The patent claimed “a process for amplifying existing nucleic acid sequences if they are present” adding “For diagnostic applications in particular, the target nucleic acid sequence may be only a small portion of the DNA or RNA in question.” Probing a whole gene isn’t necessary to identify a bacterium in spit, or a virus in blood.

As first envisioned, PCR used DNAP from E. coli, the workhorse bacterium common in our guts and in molecular biology labs. Short bits of DNA, called primers, guide the DNAP to the gene part of interest, and are labeled by incorporating a fluorescent marker. So you have to know what you’re looking for to amplify a specific gene. (One of the many gaffs in Dan Brown’s “Inferno” was using PCR to find an unknown piece of DNA, which I dissed here.)

When Mullis heated the DNA before each doubling to separate the helix halves, though, the crucial E. coli DNAP fell apart, requiring a constant fresh supply. Researchers at Cetus soon invented the first thermal cycling device, named Mr. Cycle, to automate the temperature shifts. But it was an unwieldy process, taking time.

Then Mullis had an idea: switch to a “thermostable” version of the same enzyme from Thermus aquaticus, a microbe that thrives in the hot springs of Yellowstone. Microbiologist Thomas D. Brock had discovered and described it in 1969. Bingo.

T. aquaticus is an “extreme thermophile.” It’s a member of the Archaea, one of the three domains of life along with the Prokarya (bacteria) and Eukarya (everything else). (Domains top kingdoms.)

I admired an exhibit on T. aquaticus at Yellowstone this past May, near the colorful hot springs it calls home, but was frustrated at the museum’s omission of acknowledging the microbe’s role in PCR or the importance of the Archaea in the origin of life. The info just vaguely mentions “biotech.” I told my tour group the tale of another inventor, Francis Barany, a professor at Weill Cornell Medicine. In 1991 he fell into a Yellowstone hot springs in search of a different thermostable enzyme, a ligase, burning up his leg. The enzyme picks up where a polymerase signs off, knitting the sugar-phosphate backbone of a DNA molecule to which the four types of bases attach. Dr. Barany got his enzyme and invented the ligase chain reaction.

Mullis’s magic enzyme did just fine at the high temps required to repeatedly part the DNA double helices as PCR proceeds. He and his colleagues published the retooled, much more efficient gene amplification scheme in Science in 1988.

The heat-resistant enzyme made all the difference, and soon PCR and gene amplification took off. A DNA snippet could be mass-produced to millions of copies in just hours. PCR could detect one bit of DNA in a specimen of 100,000 cells. Roche acquired the technology from Cetus in 1991, pushing it towards diagnostics.

In honor of Kary Mullis, I went in search of ever more applications of PCR and quickly came up with a new list:

• Rapid diagnostics for fungal infections (Aspergillus and Candida), TB, and hepatitis.
• Field-based PCR for acute Q fever at a combat support hospital in Iraq; for a mysterious lung infection (Chlamydophila pneumoniae) among military recruits in Turkey; and anthrax. 
• In residents of labs, zoos, and natural habitats, diagnostic tests for white nose syndrome in bats; camelpox and monkeypox; herpes in pigs, minks, monkeys, and kangaroos; and a PCR panel of four pathogens used to quarantine snakes and lizards.
• Forensically speaking, lipstick is a great source of DNA.
• PCR is used to detect pig DNA in halal cosmetics, indicating meat contamination, which is against Islam law.

Readers, please add examples!