PCR (Pilgrims and Cranberry Relish)

[En Español]

A Thanksgiving tale about DNA amplification

There are many things for which I am thankful, and I’m sure for many of us these reasons for gratitude are similar—family, friends, good health all come to mind.  But there are so many other things that make our daily lives easier and more manageable. Just to name a few: power steering, microwaves, GPS, noise-cancelling headphones, etc.

Every little discovery is important. Especially in science. Maybe now we cannot foresee its relevance, but it could be crucial in the future for the development of a great technique or cure. Working in a molecular neurobiology lab, we tackle fundamental questions about how neurons develop using different techniques in biochemistry and molecular biology. We get our hands dirty in order to discover the functions of proteins in our bodies. Science has changed a great deal in recent years and many discoveries and inventions have made our lives as scientists a lot easier. And for that I am very grateful too. For instance, I am quite sure every molecular biologist on Earth is thankful for the invention of the PCR (Polymerase Chain Reaction). Thanks to PCR paternity testing, detections of DNA mutations, DNA sequencing, cloning and other applications (in medicine, research and forensics) are possible.

The invention of PCR is also one of my favorite stories about collaboration in science (even though it was indirect and unintended). Unfortunately, sometimes not everyone who is involved in the development of a great idea is recognized for their work. So this post represents a very small tribute and a big “thank you” to those whose work goes unrecognized.

In 1969, Thomas Dale Brock and Hudson Freeze (then an undergrad and now a professor at UC San Diego) were working in Yellowstone National Park collecting bacterial samples from the geysers. Until that point, scientists believed that bacteria were not capable of surviving at temperatures above 55°C (131°F). However, Brock and Freeze were able to isolate bacteria from these samples, proving the general belief wrong. They called the bacteria Thermus aquaticus, a bacterial organism that can survive to temperatures up to 80°C (176°F). This may not appear like an exhilarating discovery to you unless you are a microbiologist. But wait…

Fourteen years later in 1983, Kary Banks Mullis, molecular biologist, chemist and surf enthusiast, invented the PCR. This is a series of reactions that would allow replication of DNA from a sample too small to be useful to perform any experiment with. So, for example, imagine you want to knit a scarf but you only have a one-foot thread of yarn. With PCR you could replicate this thread in order to generate all the yarn balls you need. Nowadays, PCR is basic for a molecular biologist in any field. PCR was originally conceived by Kjell Kleppe some years before Mullis, in 1977. He mentioned his idea in a paper where he stated that two primers (small fragments of DNA) would allow the replication and amplification of a fragment of DNA of interest [1]. However, Mullis developed the idea and improved it and that is why he was the one awarded with the Nobel Prize in Chemistry in 1993. He explains in detail how he came up with this idea in his book “Dancing Naked in the Mind Field”, an inspiring read full of other interesting (read peculiar and possibly controversial) ideas that should not be taken lightly. Many times he has claimed that the PCR epiphany occurred thanks to the use of psychedelic drugs, such as LSD.

kary

Kary Banks Mullis

“Mi little silver Honda’s front tires pulled us through the mountains. My hands felt the road and the turns. My mind drifted back into the lab. DNA chains coiled and floated. Lurid blue and pink images of electric molecules injected themselves somewhere between the mountain roads and my eyes. I see the lights on the trees, but most of me is watching something else unfolding. I’m engaging in my favorite pastime.

What subtle cleverness can I devise tonight to read the sequence of the King of molecules? (…) Yes, DNA is the big one. Tonight I’m playing with a fire that will burn as brightly as Antares, descended behind these fragrant mountains several hours ago.”

Extract from “Dancing Naked in the Mind Field” by Kary Mullis.

Mullis PCR presented a problem though. The technique needed an enzyme (a protein that improves the efficiency and speed of chemical reactions) that, unfortunately, was quickly degraded in the temperatures that reactions needed to happen. This problem forced one to top up with an enzyme every new cycle of DNA replication, making PCR not-so-efficient and quite annoying for the scientist.

How do you think this inconvenience was overcome?

Another fairly clever guy, Randall Saiki, had the brilliant idea of isolating the enzyme from Thermus aquaticus, the bacteria found by Brock and Freeze, and he re-named it as Taq polymerase. He successfully applied it in the PCR, allowing the technique to be fully operated by a machine and, more importantly, providing a useful, affordable and time-saving procedure that makes our daily lab-lives a lot easier and nicer.

But I must insist, only Kary Banks Mullis was awarded with the Nobel Prize for the PCR (shared with Michael Smith, but not with any other contributor to the development of the final PCR).

And after this brief overview about PCR, you might think that, although interesting, it is not useful for our daily lives. Well, I am afraid you are mistaken. I am sure you have seen dozens of movies and series where police show up to the crime scene to collect DNA. They make it sound like the crime scenes are soaked in DNA, but that normally is not the case. In fact, rarely does one to collect enough DNA from a crime scene to analyze and get useable results. DNA samples collected from those crimes prior to 1990 could not be amplified but thanks to the accidental collaboration between Brock, Freeze, Kleppe, Mullis and Saikis that led to PCR, DNA evidence became much more useful in solving crimes. Whatever the amount of DNA, PCR makes enough copies to analyze and compare to, for instance, DNA from the suspect. Thanks to this, many innocent people who had been at the wrong place and the wrong time were not charged for crimes they did not commit. Also, thanks to PCR we can perform paternity tests or find whether a patient’s HPV (human papilloma virus) is oncogenic.

CSI

Collecting DNA from the crime scene

In a nutshell, if Brock and Freeze had not discovered Thermus aquaticus, Saikis would have not thought of using the enzyme from the bacteria to improve Mullis/Kleppe’s idea and make PCR a super-efficient and cost-effective technique, adopted by pretty much every lab around the world. And I would not be thanking today for this wonderful and casual teamwork that led to PCR.

Reference

[1] Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG “Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA’s as catalyzed by DNA polymerases.” J. Molec. Biol. vol. 56, pp. 341–61 (1971).

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Elena Blanco-Suarez is a postdoc in Nicola Allen’s molecular neurobiology lab at the Salk Institute. She studies astrocytic-secreted factors involved in the formation of synapses, their mechanisms of release and regulation. She also collaborates in Science Education Outreach programs in the Salk Institute and Reuben H. Fleet Science Museum in San Diego.

 

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