Kary Mullis invented the polymerase chain reaction (PCR) in 1983 and was awarded the Nobel Prize in Chemistry in 1993. As an R&D nucleic acid chemist during the three decades since Mullis’ contribution, I’ve always been amazed by the ever-expanding, enabling-power of PCR. A 1998 article about Mullis in the New York Times referred to the method “virtually dividing biology into the two epochs of before PCR and after PCR.” To put the “after PCR” epoch into numerical perspective, consider the following.
- ~50,000 labs worldwide currently use PCR
- ~400,000 publications indexed to PCR currently in PubMed
- ~33,000 such publications will appear in 2013, which is…
- One PCR-indexed publication every ~15 minutes, 24/7/365 in 2013
Like PCR itself, publications related to this method keep amplifying (pun intended).
Thank you very much, Kary!
How did seeking sequencing lead to finding PCR?
If you haven’t read Kary Mullis’ witty account of “The Unusual Origin of the Polymerase Chain Reaction” published in Scientific American in April 1990, I highly recommend doing so if you can get your hands on a copy. Since this issue is not electronically available and is likely not in hard copy form in many libraries, I’ll share what I found fascinating about this story.
Freed from the toil of having to manually synthesize oligonucleotides, and instead having a machine do that for him in 1983 at Cetus, Mullis was—in his words—“puttering around with oligonucleotides” in search of “a technique for easily determining the identity of the nucleotide at a given position in a DNA molecule.” The approach he was thinking about was essentially a variation of what nowadays is called polymerase-mediated single-base extension of an oligonucleotide primer using dideoxynucleotide triphosphate (ddNTP) terminators—aka more simply “minisequencing” (see SNaPshot®). Importantly, he decided that it “would be more definitive” if some sort of minisequencing used a pair of oppositely oriented oligonucleotides—one for each strand of DNA—so as to determine the single nucleotide of interest and it’s paired base (sort of like proofreading). In Mullis’ mulling over potential problems due to trace contamination of ddNTPs with regular deoxynucleotide triphosphates (dNTPs), he suddenly recognized the essential elements of PCR: a pair of oppositely oriented oligonucleotides with any desired spacing and thermal cycling to repeatedly melt and hybridize for extension by a polymerase and dNTPs would lead to exponential amplification of any selected segment of DNA. Such an amplification method, he thought, seemed to be more useful than the original technique he was mulling over.
But here’s where the story gets even more interesting. Mullis says that “[f]or the next few weeks I described the idea to anyone who would listen [but] no one was particularly enthusiastic about it.” Undeterred, he did an initial experiment and got the expected gel result late that evening, when he bumped into Albert Halluin, the patent attorney for Cetus. Mullis told him about the idea of PCR and the results. Halluin “agreed that it was significant…[and] was even a little excited and suggested that I get to work on the experiment and write a patent disclosure. As he left he congratulated me.” So while, of course, we are forever indebted to Mullis for his discovery of the PCR process, we must also thank Halluin for recognizing the genius of the discovery.
May you Rest in Peace, Albert!
Before getting to some examples of how powerful and democratized PCR has become, I should mention a couple of free Google Book items that offer technical experience and opinions from hands-on expert-authors. One is a chapter by Innis & Gelfand entitled Optimization of PCR: Conversations between Michael and David, which I found particularly informative about original approaches to achieving “hot start PCR”. For recent improvements see TriLink CleanAmp™ Hot Start PCR products. Another is a chapter by Wittwer & Farrar entitled Magic in Solution: an Introduction and Brief History of PCR—what a clever and seemly true metaphor!
Amazing Examples of the Power of PCR
Here are just some of the many applications that underscore the transformative power—and now democratization—of PCR in the aforementioned “after PCR” epoch:
- Enable DNA/RNA library prep and quantification for next-generation sequencing (NGS) technologies (Buehler et al.)
- Enable paleogenomics (aka ancient DNA analysis); including identification of a 700,000 year old horse (Willerslev)
- Amplify long (>40-kb) fragments of DNA (Cheng et al.)
- Whole-genome amplification (Arneson et al.)
- Single-cell gene expression (Fluidigm)
- Chemically synthesized bacterial genome (~1-Mbp) (Gibson et al.)
- Single-tube 6,144-plex amplification of 10ng DNA (Leamon et al.)
- Ultra high-throughput (230,400 reactions/run) tape format (Douglas Scientific)
- Digital PCR quantitation formats; e.g. 10 million pL-sized droplets per lane (Raindance).
- Ultra-fast (<3 minute) amplification (Wheeler et al.)
- Semiconductor-based pH detection of PCR (DNA Electronics)
- Selection of DNA/RNA aptamers (TriLink)
- Low-cost ($599 via Amazon) OpenPCR thermal cycler for “Do-It-Yourself” (DIY) Biologists
- PCR for the high school biology classroom (UConn)
- “Pocket PCR for Pennies” (LavaAmp)
- Solar powered microfluidic PCR devices for point-of-care diagnostics (Jiang et al.) (See image below)
In closing, I hope that you are impressed by the power of PCR. For me, the “magic moment” for experiencing the power of PCR came during an NGS DNA methylation study when I did my first-ever single-molecule PCR amplification. As a chemist, the notion of doing anything with a single molecule of anything, let alone DNA, seemed impossible—but it wasn’t—it was actually ridiculously easy, thanks to Kary and the 30 years of PCR innovation that he inspired. Here’s to 30 more years!
As always, your comments are welcomed.
By the way, depending on how fast or slow you read this blog and how many links you checked out, at least one more PCR paper was published—amazing!
What is Kary Mullis doing now?
According to www.karymullis.com, which has lots of interesting content, Kary Mullis’ company—Altermune LLC—is investigating “chemically programmable immunity” using conjugates of antibodies and aptamers for targeted therapies. Altermune’s stated strategy for helping the immune system is to “[g]ive its antibodies, its workhorse molecules, bionic arms, little chemical extensions that allow an old antibody to do new tricks.” Check out Mullis’ patent application for details.
Enabling the Enabler
Ready access to synthetic oligodeoxynucleotides (ODNs) gave Kary Mullis the opportunity for “puttering around” with them, which led to his inventing PCR that in turn enabled many applications. Since every PCR reaction requires a pair of ODN primers, it’s fair to view ODNs as “enabling the enabler.” Just as PCR has led to the “after PCR” epoch, automated ODN synthesis has led to the “after ODN” epoch. Try to think of what health-related sciences would be like if ODNs weren’t available. Fortunately, that’s just a mental exercise, thanks to Marv Caruthers, Applied Biosystems Inc. (ABI) and others whose collective work engendered the “after ODN” epoch—some of which will be the content of my future blogs later this year. Stay tuned.
Oh, one more thing. I tried to find a good PCR cartoon using Bing Images and Google Images, but this is the only one that I thought was worth sharing—primarily because it lets you know that there’s a website called www.biocomicals.com that you can visit during a coffee break. It has a readers map that seems to show more readers are in Europe than anywhere else—I wonder what that means? If you have any good PCR cartoons to share, I’d love to see them! Please post them in the comments section.