World’s Smallest Real-Time PCR Device

  • Fits in the Palm of your Hand, and Has Single-Molecule Sensitivity
  • Analyzes 4 Samples, and Can be Modified to do 8 
  • Project Leader Reveals Commercialization Details 

I think we’re all fascinated by catchy headlines touting the world’s biggest, tallest, etc., so a recent publication by Ahrberg et al. in venerable Lab on a Chip claiming the world’s smallest real-time PCR device instantly struck me as blogworthy. It also seemed quite apropos as a follow-up to my previous blogs on the continuing shrinkage, so to speak, of real-time PCR technology for point-of-care qPCR diagnostics or other emerging applications in the field.

This hand-held real-time PCR device, developed by A*STAR Singapore, is amazingly small in comparison to the first real-time PCR system introduced by Applied Biosystems in 1995 that weighed 350 pounds and had a width of 7 feet, thus requiring an entire bench top.

pcr

Left: World’s smallest real-time PCR device. (Taken from Ahrberg et al). Right: Applied Biosystems 7700 real-time PCR system. (Taken from distrobio.com).

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Norovirus: Science Behind the Headline

  • The Virus is Quite Common with 267 Million Cases and 200,000 Deaths Annually
  • RT-PCR is the Detection Method of Choice
  • First Cell-Culture System May Speed Drug and Vaccine Development

We’ve all seen TV news stories about disgruntled passengers disembarking cruise ships returning to port early because of an outbreak of nasty gastroenteritis (i.e. inflammation of the stomach and intestines leading to nausea, vomiting, diarrhea, and stomach cramps). Norovirus (NoV) is the causative agent of these frequently reoccurring “nightmare” cruises, of which 13 have been reported since 2012, sickening some 200-600 passengers. It’s not just limited to cruises, the virus affected 100+ students at a school in Eugene, Oregon last year. And now there’s new evidence for transmission of NoV by eating oysters—which I will therefore not eat in the future.

Taken from counselheal.com.

Taken from counselheal.com.

But perhaps the most NoV-related media attention—and investor ire or litigant action—has been recently focused on gastroenteritis outbreaks at Chipotle—a popular restaurant chain. A criminal investigation is under way at Chipotle, and according to an Associated Press report the company has been served with a federal subpoena.

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‘Genospirituality’—In the Spirit of the Holidays

This last blog for 2015 comes at a time when many of us are looking forward to the upcoming holiday season to enjoy, in various ways, being or sharing with family and friends, and reflecting thankfully for what we have. We often refer to this as getting into “the spirit” of the holidays, regardless of one’s religious or secular beliefs.

In this context, and with the nucleic acids research-relatedness of my blogs in mind, I thought it would be apropos to tell you a bit about some intriguing research aimed at assessing genes associated with spirituality, by which is meant “an inner search for enlightenment achieved through practices such as prayer [religious] or meditation [secular]”, as elaborated elsewhere.

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DNA Barcodes to Follow Food and Foil Fraud

  • Fork-to-Farm Traceability Proven Practical
  • Uses qPCR Decoding of DNA Barcode “tags”
  • Consumer Concerns May Curb Commercialization

In earlier blogs here I’ve highlighted how sequencing of DNA barcodes has been used to identify fraudulent food sales—such as cheap meat or fish passed off as expensive steak or sashimi—and the same for mislabeled herbal products. Now, I’d like to share with you some nifty work by researchers who have applied qPCR to detect DNA barcode “tags” to follow foodstuff from your fork back to the farm (or farms) from which it originated.

This is not a simple goal to achieve, scientifically, and would certainly have been exceedingly useful, societally, in tracing the origin of food-related incidents such as listeria-contaminated cantaloupes that led to 29 deaths several years ago. In fact, just a few weeks ago there was a deadly outbreak of salmonella linked to cucumbers imported from Mexico wherein 4 people died and 732 were sickened by the bacteria.

They all look the same, but sometimes it’s very importatnt to find out from where they came. Taken from en.wikipedia

They all look the same, but sometimes it’s very important to find out where they came from. Taken from en.wikipedia

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Point-of-Care PCR 2.0

  • Ubiquitome Quickens Pace of POC Apps for Its Freedom4
  • Cepheid Unveils its POC Diagnostics System
  • Hopkins Crew Brews “Coffee Mug-Sized” Gizmo for Fully Automated Chlamydia Testing
Kiwi Dr. Jo-Ann Stanton holding Ubiquitome’s Freedom4 at Tri-Con 2015

Kiwi Dr. Jo-Ann Stanton holding Ubiquitome’s Freedom4 at Tri-Con 2015

Regular readers of this blog will recall a recent byline exclaiming “Honey I Shrunk the qPCR Machine”, which spotlighted the unveiling of startup company Ubiquitome’s first point-of-care (POC) product—Freedom4—developed in New Zealand. Up until then, this far away—for me—exotic island country brought to mind folks fondly nicknamed Kiwi—after the native flightless bird, not Chinese fruit. Mightily impressed by this tiny but powerful qPCR device, I vowed to thereafter keep an eye on these Kiwis’ democratized POC apps enabled by its nifty handheld 4-sample high-performance qPCR device.

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Gene-Expression Biomarkers Can Detect Depression

  • First-ever Lab Test for Depression Found Using RT-PCR
  • FDA Approval as Diagnostic Possible by Early 2016
  • Huge Potential Market as 1-in-10 US Adults Suffer from Depression

While it’s normal for everyone to occasionally feel blue or sad, prolonged bouts of depression that interfere with normal life are indicative of a serious mental health issue. While there are numerous forms and differing severity of depressive disorders, as described at a National Institute of Mental Health (NIMH) website, only two factual aspects of this illness really stand out in my opinion:

Redder countries have higher depression rates. Bluer countries have lower depression rates. Taken from The Washington Post.

Redder countries have higher depression rates. Bluer countries have lower depression rates. Taken from The Washington Post.

  • Depression is a very common illness. The Centers for Disease Control and Prevention estimates that 1-in-10 US adults suffer from depression, which reportedly costs close to $50B annual in lost productivity in the work place. Globally, more than 350 million people of all ages are afflicted with depression, according to recent statistics from the World Health Organization (WHO). By the year 2020, WHO estimates that depression will be the second leading cause of “lost years of healthy life”, following heart disease. Incidentally, as seen from the map below, depression rates around the world vary significantly among countries.
  • Depression is diagnosed based on the patients’ self-report of their symptoms and the evaluation of one or more structured psychiatric interviews with the patient by a psychiatrist, psychologist or primary care physician. The absence of direct, non-subjective measures of depression can lead to relatively lengthy time-to-treatment, non-reporting, or—sorry to say—fraudulent claims and/or treatments based solely on what is said as opposed to what is objectively measured.

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Top Picks from Tri-Con 2015

  • “Honey I Shrunk the qPCR Machine” Tops Presentations
  • High School Student Wins Popular Vote for Best Poster
  • BioFire Defense FilmArray is More Interesting Exhibitor
  • Extra Bonus: Swimming with the Sharks

The 22nd International Molecular Medicine Tri-Conference—better known as Tri-Con—took place on Feb 15-20 in San Francisco, where I and 3,000+ other attendees from over 40 countries took part in a jam-packed agenda. In this blog I’ll briefly share my top 3 picks—and an “extra bonus”—but first some insights into the challenges involved in navigating a large conference like this.

The first challenge was scoping out four simultaneously occurring “channels”—diagnostics, clinical, informatics, and cancer—to select as many interesting items as possible from all the presentations (500), panel discussions (30), posters (150), and free “lunch-nars.” The new Tri-Con’15 app with a word and name-searchable agenda (including abstracts) made this easier than previous years. I was even able to put selected items into a calendar/to-do list with 15-min reminder alarms—very slick and convenient. Every big conference should have an app like this!

The second challenge came once I was physically onsite. It took a bit of effort to navigate from one room to another in the huge, multi-room Moscone Center without GPS guidance. I was also struggling to make it to the talks and events on time without getting hijacked by bumping into friends—which happened a lot.

The third and final challenge had to do with posters. Given all of the other exciting options during the conference, I really had to focus to stay on-task and make sure I was present at my poster at the specified times, yet alone try to get around to the other posters of interest. This was definitely not easy, since my poster entitled Pushing the Limits of PCR, qPCR and RT-PCR Using CleanAmp™ Hot Start dNTPs attracted a steady stream of interested visitors. But that’s a great challenge to have, so I can’t complain too much.

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Children at Risk from Deadly Respiratory Virus EV-D68

  • Frightening Statistics From CDC
  • CDC Updated U.S. Map of Outbreak & Advice of What to be Aware
  • CDC Develops Rapid Real-Time RT-PCR Test for Detection
  • Some Speculate on Linking Outbreak to the “Southern Border Invasion”

Ebola virus is dominating news reports lately, and perhaps rightly so considering the worldwide impact. Turning our attention, however, to actual incidents of infection and death in the U.S., enterovirus (EV) D68 poses a much greater threat and warrants our attention—especially if you or your friends have young children.

On September 24, Eli Waller’s parents were worried that their 4 year-old son had pink eye and kept him home from school so that he wouldn’t infect other children. He seemed otherwise healthy. What happened next was shocking.

Eli Waller (Credit Andy Waller, via Associated Press). Taken from NY Times.

Eli Waller (Credit Andy Waller, via Associated Press). Taken from NY Times.

‘He was asymptomatic and fine, and the next morning he had passed,’ said Jeffrey Plunkett, the township’s health officer. ‘The onset was very rapid and very sudden,’ quoted the NY Times.

A week later the Centers for Disease Control and Prevention (CDC) confirmed that Eli had been infected with EV-D68.

EV-D68 was seen as early as August of this year as hospitals in Missouri and Illinois reported increased visits from children with respiratory illness. Soon, the virus was identified in 43 states and detected in 594 patients, 5 of which died.

After reading this very sad—if not frightening—story, I decided to research EV-D68 for this “hot topic” blog, which I’m dedicating to little Eli Waller.

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PCR Better than Pap Test for Preventing Cervical Cancer

“Power of PCR” as a Transformative Diagnostic Method

  • FDA Approves Roche PCR Test for Cervical Cancer Screening
  • Automated Test Replaces Pap Test as First-line Cervical Cancer Screening
  • Demonstrates the “Power of PCR” as a Transformative Diagnostic Method

Pap Test

The Papanicolaou test—aka Pap test, Pap smear, cervical smear, or smear test—is a method of cervical screening used to detect potentially pre-cancerous and cancerous processes in the endocervical canal of the female reproductive system. Unusual findings are often followed up by more sensitive diagnostic procedures, and, if warranted, interventions that aim to prevent progression to cervical cancer.

Proper interpretation of microscopic results requires a “trained eye.” This is evident from the representative example shown below, which I found in an online educational textbook and, quite frankly, had trouble discerning the visual keys described in the verbatim caption. Notwithstanding this issue, Pap tests were—until now—the accepted “gold standard.”

pap

Taken from homepage.smc.edu via Bing Images.

The source document for this Pap smear reads as follows. “The cytologic features of normal squamous epithelial cells can be seen at the center top and bottom, with orange to pale blue plate-like squamous cells that have small pyknotic nuclei. The dysplastic cells in the center extending to upper right are smaller overall with darker, more irregular nuclei.”

The eponymous test was pioneered by Georgios Papanikolao, a prominent Greek doctor, who in 1928 was the first to report that uterine cancer could be diagnosed by means of a vaginal smear. However, the importance of this work was not widely recognized until his 1943 publication of Diagnosis of Uterine Cancer by the Vaginal Smear, coauthored by Herbert F. Traut, both at Cornell University Medical College.

ResearchGeorgios Papanicolaou moved to Miami, Florida in 1961 to establish the Papanicolaou Cancer Research Institute at the University of Miami, but died in 1962 prior to its opening. Papanicolaou was the recipient of the Albert Lasker Award for Clinical Medical Research in 1950—this award is sometimes referred to as “America’s Nobels,” as eighty-six Lasker laureates have received the Nobel Prize. Papanikolaou’s portrait appeared on the Greek 10,000-drachma banknote of 1995-2001, prior to its replacement by the Euro.

Cervical Cancer Statistics

Cervical cancer is the second most common cancer in women worldwide, according to an NIH publication in 2007. Country-by-country data for cervical cancer reveal a striking geographical distribution. According to currently available U.S. Centers for Disease Control (CDC) FastStats, cervical cancer mortality in the U.S. in 2010 was ~4,000 or ~2.5 deaths per 100,000 females.

The global statistics provided by Cancer Research U.K. are far more saddening. Worldwide there were more than ~275,000 deaths from cervical cancer in 2010 that accounted for ~10% of female cancer deaths.

Remarkably, mortality rates are reported to vary seventeen-fold between the different regions of the world. By estimating the years-of-life-lost (YLL) by young and middle-aged women (25-64 years old) in different regions of the world, YLL attributed to cervical cancer is the most important cause of YLL for all cancers in Latin America, the Caribbean, and populous regions of Sub-Saharan Africa and South-Central Asia. The overall picture is not very sensitive to the age-weighting function used. The report notes that, since this loss of life is preventable using existing technologies, more health-resource allocation in low income settings is needed.

Pap Test Statistics

Currently available CDC FastStats for Pap test use in the U.S. in 2010 (the most recent year available) are as follows:

  • Percent of women 18 years of age and over who had a Pap test within the past 3 years: 73.2%
  • Number of physician office visits during which Pap tests were ordered or provided: 29.4 million
  • Number of hospital outpatient department visits during which Pap tests were ordered or provided: 2.4 million

Pap Test Recommendations as of 2013

To put today’s blog-post headline about switching from Pap to PCR in perspective, here are snippets from the most recent CDC guidelines and comments made available in a January 2013 press release headlined with “more women getting Pap tests as recommended [but] some women get Pap tests without need.”

  • In 2012, the U.S. Preventive Services Task Force, American College of Obstetricians and Gynecologists and American Cancer Society recommended that women, beginning at age 21, should start Pap test screening every three years.
  • The same groups agree that screening is unnecessary for most women who have had a total hysterectomy (removal of the uterus and uterine cervix) for non-cancerous reasons, or for women aged 65 years and older with several years of normal test results.
  • Studies analyzed Pap test survey data from CDC’s Behavior Risk Factor Surveillance System found the following:
    • The percentage of women aged 18-21 years who reported never being screened increased from 23.6% in 2000 to 47.5% in 2010; however, screening is not recommended for women under the age of 21.
    • In 2010, 58.7% of women aged 30 years and older who had a hysterectomy were still given a Pap test.
    • Because of the Affordable Care Act (aka Obamacare), many private health plans and Medicare now cover certain preventive services, including cervical cancer screening, with no copays or other out-of-pocket costs.

HPV: The Cervical Cancer-Causing Agent and Key to Early Detection

In a landmark publication in 1999 entitled Human papillomavirus is a necessary cause of invasive cervical cancer worldwide, Dutch investigators used PCR data to establish that the worldwide HPV prevalence in cervical carcinomas is 99.7 per cent. They noted that “the presence of HPV in virtually all cervical cancers implies the highest worldwide attributable fraction so far reported for a specific cause of any major human cancer.” More importantly, they presciently concluded that “the extreme rarity of HPV-negative cancers reinforces the rationale for HPV testing in addition to, or even instead of, cervical cytology in routine cervical screening.”

Due in part to technical challenges posed by numerous genotypes of HPV with varying cancer causality detailed elsewhere, and unavoidable time-consuming clinical studies required for FDA approval, it has taken ~15 years for a PCR test to now be poised to displace the Pap test as the primary diagnostic approach for early detection of cervical cancer.

Those of you who are interested in the technical underpinnings of Roche’s investigations to this end are referred to this 2013 publication by Roche and collaborators entitled Development and characterization of the cobas human papillomavirus test. In contrast to the tedious Pap test protocol and its “visually challenging” manual microscopic analysis, this “cobas”-based PCR test provided by Roche is fully automated.  The test process involves two instruments: one that completes sample preparation (COBAS® AmpliPrep) and another that performs the PCR process and detection of the pathogen DNA in real time (COBAS® TaqMan® Analyzer).

Incidentally, I traced-back the term “cobas” to late 1970’s Roche instrumentation named the “cobas-bio” analyzer, but could not decipher what “cobas” stands for! If any of you know the answer, please let us know by a comment at the end of this post.

FDA Panel Recommends Replacement for the Pap Test

This attention-grabbing headline of a March 2014 NY Times article by Andrew Pollock was the catalyst for my decision to research and write this blog exemplifying the “power of PCR” as a transformative diagnostic method. While this and numerous other popular news media all made reference to an FDA panel’s report, it took some digging to find the actual source-report, which is an 80-page pdf that can be accessed here to peruse in detail, if you wish. However, a much shorter but essential-fact-laden article by Joyce Frieden, News Editor of MedPage Today provided the following excerpts.

The FDA’s Medical Devices Advisory Committee Microbiology Panel agreed by a vote of 13-0 in each of three successive votes that the cobas® viral DNA test for HPV—made by Roche Molecular Systems—was safe and effective for cervical cancer screening, and that the benefits of the tests outweighed the risks. The Panel recommended that this Roche HPV test replace the Pap smear as the first-line standard of care for cancer screening.

The Roche test is seen as better than Pap tests in finding precancerous lesions (taken from the NY Times).

The Roche test is seen as better than Pap tests in finding precancerous lesions (taken from the NY Times)

The cobas® test currently has approval as a follow-up assessment for women 21 and older who have abnormal Pap tests, and as a co-test with the Pap smear to screen for the high-risk p16 and p18 HPV strains in women 30 to 65. The test comprises genotyping for HPV16 and 18 and pooled assessment of 12 additional high-risk HPV strains.

According to the proposal submitted by Roche, women 25 and older who test positive for HPV16 or 18 would proceed directly to colposcopy for further assessment.

Patients who test negative for HPV16 or 18 but positive for the other high-risk strains would have a Pap test to determine the need for colposcopy. Women who have a completely negative test would be followed at their physician’s discretion.

Panelists did express some concerns about dropping the age at which women should have the test from 30 to 25. The ATHENA study of over 47,000 patients with long-term follow-up used as the basis for the application found that about 11% of women ages 25 to 29 tested positive for HPV16 or 18 with the cobas test, compared with 7.28% among women 25 to 29 who had cytology alone as their first-line screening. Panel member Paula Hillard, MD, of Stanford University in California, was quoted as saying that would mean more patients in that age group “will be anxious about potentially having cancer.”

In addition, Hillard is quoted as expressing concern about off-label use. “I’m concerned that all those women potentially with other high-risk positivity won’t go to Paps next but go [straight] to colposcopy. That’s not what’s proposed here, but what control does FDA have once it’s out there?”

Panelist Kenneth Noller, MD, of the American Board of Obstetrics and Gynecology, in Dallas, agreed that real-world use could differ from the protocol proposed by Roche. He’s quoted as saying that “I’ve been watching how people practice; if you’re high-risk HPV positive you’re going to get colposcopy.” Furthermore, he said “that doesn’t necessarily mean it’s bad—it’s what you do with the colposcopy.”

Noller added that although he was “somewhat biased against dropping the age to 25 before I came here … I find the data presented today somewhat compelling to drop it to 25.”

Agreeing with this was panel member Kimberly Hanson, MD, MHS, of the University of Utah and ARUP Laboratories, both in Salt Lake City: “now we have the opportunity to identify women earlier, and to me that’s compelling,” adding that “although colposcopy is invasive and can be anxiety-provoking, it’s really very safe, so I think I’m leaning toward earlier screening.”

According to the summary submitted by FDA staff members, “The data show that the proposed primary screening indication for the cobas HPV test detects more women with disease and requires fewer women without disease to go to colposcopy than cytology alone.”

Benefit-risk analyses favored the HPV DNA test whether expressed in terms of number of cases of high-grade cervical disease per 10,000 women screened or per 100 colposcopy procedures.

The FDA is not bound to follow its advisory committees’ recommendations, but does so in most cases. On April 25—coincidentally DNA Day 2014—the FDA formally approved Roche’s HPV test as the First-Line Cervical Cancer Screening Method.

The “Entrenchment Factor”

At the risk of “throwing cold water” on the aforementioned PCR test benefits, I feel compelled to quote from Pollak’s NY Times story that ended with the following caveat.

“The Pap test, which is well entrenched and has been highly successful, will not go away quickly, if at all, however.

Assuming the FDA itself agrees with its advisory committee and approves the new use of Roche’s test, it would become just another option, not a replacement for the older testing regimens. And many doctors will not adopt the new test unless professional societies recommend it in guidelines, which could take years.”

Let’s all hope that these professional societies—and any other persuasive factors—lead to relatively rapid adoption by doctors.

As always, your comments are welcomed.

30 Years of PCR and Going Strong!

Amazingly, there’s one PCR publication every ~15 minutes, and it’s still getting faster, better, cheaper and more enabling! Human DNA

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!

Kary Mullis

Kary Mullis (Bing Images)

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)

pcrsolar

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!

Postscript 

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.

Bio-Humor

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.

itsPCRtime

Image from BioComicals