Interviewer: Lydia Morrison, Marketing Communications Writer & Podcast Host, New England Biolabs, Inc.
Interviewee: Gregory Patton, Development Scientist III, New England Biolabs
Hello and welcome to the Lessons From Lab and Life podcast. I'm your host, Lydia Morrison, and I hope that our podcast offers you some new perspective. Today I'm joined by NEB development scientist, Greg Patton, who's going to tell us all about how PCR is being used today, both inside and outside the lab.
Today I'm joined by Greg Patton, an applications and product development scientist at NEB. Thanks for taking the time to talk to me today Greg.
Thanks for having me.
Can you give us a brief overview of PCR?
As a quick summary, the polymerase chain reaction or PCR for short, was a molecular biology technique invented in 1983 by a tenacious scientist named Kary Mullis. That was over 30 years ago. So obviously quite a lot has changed. What hasn't changed is the fundamental principle of PCR. At its core PCR is used to exponentially amplify a specific sequence of DNA, creating billions of copies of that sequence of interest when potentially only a few copies were originally available. Amplification requires an enzyme known as a polymerase and a thermocycler, which is just a fancy heat block that cycles between various temperatures required for DNA synthesis.
PCR is a powerful tool for determining the presence or absence of a particular sequence of DNA, making it a great qualitative way to attain a simple yes, no diagnostic answer. There have been numerous modifications of the technique over the years creating variants of PCR to extend its use in research, forensic science, medicine, and agriculture.
So, can you give us an example of where PCR impacts our daily lives?
Sure. When you grab a burger at a fast food restaurant or purchase chicken wings at your local grocery store-
Yes, exactly. You assume that these foods are safe to eat. That assumption is generally a good one due to the number of food safety practices in place to keep everyone safe from nasty foodborne pathogens. Our foods in the manufacturing processes, they undergo or actively monitored by a number of scientific techniques including PCR.
How does that work?
Foodborne pathogens can be identified by PCR because these pathogens have unique DNA signatures and the signatures can be amplified and detected, quickly giving a yes, no answer about potential contamination. Of course, nothing is perfect, especially given the number of steps in our food production chain. The result is 48,000,000 cases of foodborne illness annually according to the Center for Disease Control and prevention, or just better known as the CDC.
Pathogenic organisms can hide in beef, in eggs, spinach, cheese, berries, and the list goes on. Because the bad guys can hide in raw eggs and uncooked flour, you should never eat raw cookie dough. But between you and me, it's too delicious to resist sometimes.
I couldn't agree more, but it is scary. I frequently see food recalls and outbreaks of foodborne illness on the news or in my social media newsfeed.
Yeah. One of the most memorable foodborne illness outbreaks in recent years in the United States occurred back in 2015 and involved in outbreak of E. coli (STEC) 026 at a popular Mexican chain restaurant. The CDC indicated that 55 people felt ill over 11 different states. Luckily in this case, there were no deaths reported. Unfortunately, this wasn't the case back in 1993 when undercooked hamburgers at a fast food chain caused hundreds of ill customers in Washington, California, Idaho, and Nevada. Tragically, four children died due to eating meat containing the dangerous E. coli 0157 strain. It was this outbreak that lead to stronger regulations regarding the handling of our food.
But how does the CDC monitor that?
Pinpointing the contaminated food source and identifying the pathogens responsible for an outbreak is necessary to prevent further illness from spreading. Detection of outbreaks relies on our public health surveillance system, which utilizes PCR to identify and diagnose pathogens causing illness in the general public. Stool or other biological samples are taken from patients who are suspected of having food borne illness. These samples are analyzed by a number of analytical methods including PCR to determine the exact pathogen responsible for their illness.
Individuals affected by identical pathogens can be linked, and then these clusters of illness can ultimately provide a roadmap back to the source of contamination such that further illness can be prevented. Providing a safe food production chain so that the foods you consume on a daily basis are safe to eat, is just one manner in which PCR plays a behind the scenes role in our everyday lives.
So can you tell us a little bit more about how PCR is being used to diagnose illness?
The very first clinical diagnostic PCR assay was released by Roche Diagnostics systems in 1993. The Roche Amplicor PCR assay was used for the detection of the bacterial pathogen chlamydia trachomatis, the most common sexually transmitted infection for which treatment is available. The test amplified a unique DNA sequence found in a 7.5 kilobase of cryptic plasmid, which is just a piece of DNA of unknown function in the bacteria. It is present in a majority of chlamydia isolates. If you visit a healthcare provider and a suspected infection, they may swab the infection site or ask for a urine sample.
Does it matter how the sample is collected?
Well, they're just trying to isolate some of the offending pathogen, in this particular case we're talking of bacteria. No matter the sample collection method, the goal is detection and identification of the bacteria causing illness. Bacterial infections have historically been diagnosed by a bacterial culture, which basically means the bacteria are grown in a controlled laboratory setting until there is a sufficient quantity to make a clear diagnosis. It's the gold standard method of detection and diagnosis even today.
So how long does that take? Because I remember my daughter was ill once and she had a swab taken and they were growing up a culture for that and I think it was like two to three days before we heard back about whether or not she could be treated with antibiotics.
Well, exactly. Bacteria often grow at extremely slow rates. So the bacteria culture suffers from very slow turnaround times, delaying proper diagnosis of patients for up to several days. PCR assays in contrast, allow results to be attained in just a few hours. Fast sample to answer times improves patient care by allowing the correct treatment to be as prescribed earlier by a healthcare provider. This can reduce the spread of infections to other individuals and in some cases, has lifesaving consequences.
So how do healthcare providers know which test should be performed?
Well, your symptoms play a big role obviously. But often multiple pathogens can be evaluated simultaneously. One of the advances of PCR since its invention is the ability to multiplex. Instead of using one single amplification reaction to detect one sequence of DNA, multiplexing permits detection of multiple sequences of DNA in a single reaction resulting in multiple simultaneous yes, no answers regarding infections. In the case of sexually transmitted infections, diagnostic multiplex PCR tests often evaluate the presence or absence of several major pathogens such as chlamydia and gonorrhea using a single sample. Multiplex detection reduces the overall assay cost, increases testing through put and limits patient inconvenience.
PCR detection is also not limited to just bacteria. It is used to detect other pathogenic organisms such as viruses, fungi and protozoa.
Are there advantages to PCR as a diagnostic tool other than the fast sample to answer time and the ability to multiplex?
Not only can PCR detect DNA, but it can also be used to interrogate RNA as well. RNA is the intermediate and the central Dogma of Biochemistry that states that DNA makes RNA, which then makes proteins that carry out specific functions in living organisms. RNA is just a biological polymer, much like DNA and has a variety of functions in the cell. By coupling an enzyme known as a reverse transcriptase upfront of the PCR step, the information encoded RNA can be converted back to DNA and then amplified. This process is better known as a reverse transcription PCR or RT-PCR, and that shouldn't be confused with real-time PCR, which I'm sure it we'll touch upon later.
Also, given that PCR can detect just a single copy of DNA, it is a highly sensitive technique, meaning infections can often be detected earlier and more accurately when compared to conventional clinical diagnostics. Detection of RNA makes PCR particularly useful in identifying RNA viruses like influenza, HIV, and Zika.
So how exactly is PCR and point-of-care testing (POTC) use to detect, say HIV?
Well, in the case of HIV, the current gold standard method of detection is combined antibody and p24 antigen based detection, and it's often referred to as fourth and fifth generation HIV testing. Antibodies are essentially proteins the body produces and response to infections. The problem with antibody based testing is that it makes many weeks for our immune system to produce antibodies in response to the virus. Meaning antibody based testing alone cannot detect recently infected individuals. Therefore, antibody testing is coupled with a p24 antigen test which looks directly for the virus and can detect acute infections approximately two weeks after being infected. The combined antibody antigen test takes only a few hours to complete and allows for accurate diagnosis of HIV infections in most but not all individuals.
So which individuals would this method not work for?
In some situations, such as testing of infants and children less than 18 months, nucleic acid tests such as PCR or RT-PCR are superior and are therefore performed instead. This is especially true in the case of babies born to HIV infected mothers. This is because maternal antibodies are passed onto newborns at birth and therefore an antibody-based test is not an accurate way to establish infection in young children.
Because you would have like a false positive?
Yes, correct. Furthermore, the p24 antigen test is less sensitive than PCR in the first few months after birth. PCR helps accurately establish infections in newborns such that proper HIV treatment can be started immediately. Another advantage of RT-PCR over antibody-based HIV testing is that RT-PCR can be used to monitor the viral load or the amount of HIV RNA in an infected individual overtime. By detecting the amount of HIV RNA present in the body, RT-PCR can determine how effectively antiviral treatments are controlling the virus.
Wow, that's amazing. So does RT-PCR only allow physicians insight into which antiviral treatments to treat with, or can it also be used to measure the efficacy of a treatment that's currently being administered?
It helps with both really. HIV is treated with drugs known as antiretrovirals or ARVs. These are drugs that are always given in combination, often referred to as an antiretroviral therapy. RT-PCR and other laboratory tests are used to simultaneously guide which combination of antiretrovirals will be most effective before initial treatment and during therapy.
You mentioned that RT-PCR shouldn't be confused with real time or quantitative PCR. How is quantitative PCR different from typical PCR?
Quantitative PCR or qPCR is just PCR coupled with a way to monitor the reaction. Unlike traditional PCR, which is analyzed upon reaction completion, determine the presence or absence of a DNA sequence. qPCR monitors DNA amplification as the reaction progresses, or in real-time. qPCR not only gives a yes/no answer, but also tells you the relative amount of DNA that's present in any given sample. There are several ways to monitor amplification. But one of the most common methods is a DNA and intercalating dye. A dye that binds a double stranded DNA and gives off a fluorescent signal that can be measured with an optical detector.
Exponential amplification in PCR means that the amount of DNA doubles at every PCR cycle, which leads to an increased amount of bound intercalating dye, and thus a greater fluorescent signal. qPCR can be semi quantitative by comparing the amplification signal generated between two different samples or it can allow for absolute quantification by comparing a sample to a set of standards of known concentration.
Mm-hmm (affirmative). I see. So, can you give us an example of how qPCR is used?
There are two big applications of qPCR that come to mind. qPCR is often used to detect copy number variations. In general, people have two copies of most genes, one copy inherited from the mother and the other inherited from their father. Deviations from two copies can occur due to duplications, deletions or insertions in our genomes. qPCR is helping us understand how differences in gene copies impact human health.
The second application, which is probably the most prominent application, is gene expression analysis. It requires coupling a reverse transcriptase upfront of qPCR to enable RT-qPCR. Gene expression is the process by which the information encoded in DNA is turned into RNA, which then results in functional products within the cell. RT-qPCR is enabling a better understanding of this highly regulated process and how gene expression imbalances play a role in certain diseases such as cancer.
In terms of quantification, there seems to be a good deal of interest in digital PCR as well. What is digital PCR?
Digital PCR or just dPCR, allows for absolute quantification, meaning standards of known concentration are not required, like in qPCR. dPCR relies on splitting a typical amplification reaction into multiple smaller reactions. Partitioning amplification reactions into separate oil droplets in water is probably the most common format of digital PCR and is better known as droplet digital PCR or ddPCR. When the sample is split into thousands of tiny oil droplets, only a portion of those droplets end up containing DNA target. Even though each droplet contains all components required to support amplification. Commonly the goal is to attain individual reactions that contain either zero or one copies of the template DNA.
Amplification is detected in a similar manner to qPCR, by checking the fluorescence signal in each droplet. If a droplet contains the target sequence, it will generate a fluorescent signal. If the droplet lacks the template sequence, no signal is detected. The fraction of positive droplets to negative droplets is analyzed using statistics known as Poisson statistics to determine the concentration of DNA present in the original sample. Analyzing individual template molecules from a sample and droplets rather than a pool of all the molecules in a single reaction allows for greater sensitivity. Therefore, dPCR is extremely useful in identifying a small number of mutated DNA molecules in a sea of normal DNA molecules, which is often the case where certain diseases such as cancer.
So as the advantage of ddPCR, that you're using a single copy of DNA to generate this amplification?
Well in theory, all PCR types can amplify a single copy of DNA and using the power of statistics allows it to have much greater sensitivity.
How has digital PCR being used?
Digital PCR is frequently used to detect and monitor certain cancers through a non-invasive process known as liquid biopsy. Liquid biopsy is the sampling of biologic fluids, often blood and analyzing those samples for signs of disease. Tumor status can be monitored using liquid biopsy and dPCR because cancer cells shed tumor related DNA fragments known as circulating tumor DNA, into the bloodstream. However, the amount of normal circulating free DNA from healthy cells greatly exceeds the number of tumor DNA fragments. Detecting this low abundance tumor DNA allows healthcare providers to determine the effectiveness of cancer treatments, track changes into her status, and monitor patients in remission.
Unfortunately, there are limitations of dPCR in this particular application. The genetic mutations responsible for the cancer must be known prior to investigation and only a limited number of cancer mutations can be analyzed simultaneously.
So it would be impossible to pick up a novel cancer mutation using digital PCR?
What other applications require detection of cell-free DNA?
Another exciting field that relies on cell-free DNA detection is non-invasive prenatal testing. Fetal DNA is shed into the mother's bloodstream and can be detected approximately two months into pregnancy. A simple blood draw can then be used to check for genetic abnormalities of the fetus. A combination of PCR and next generation sequencing often referred to simply NGS, can then be used to determine the risk of having a baby born with specific genetic conditions.
So I've heard about PCR and NGS before. Can you tell us a little more about how PCR is used with next generation sequencing?
NGS is a high throughput sequencing technology that has absolutely exploded in recent years. NGS determines the exact sequence of building blocks that make up DNA and RNA. Many NGS workflows require amplification of DNA fragments by PCR in order to have enough material to be able to sequence and attain the genetic information. The combination of these technologies has greatly increased our knowledge of the world around us. One of the stories that I often tell people that involves NGS and PCR is the story of Nicholas Volker.
Dangerous intestinal issues, plague Nick as a young child. He underwent over 100 surgeries including the removal of his colon. His disease remained undiagnosed until doctors decided to amplify sequence and analyze his genome. A single mutation was identified that was responsible for his illness, and doctors finally identified a method to treat his disease. They performed a transplant with cells isolated from umbilical cord blood.
While Nick lives with complications that stem from his time battling the disease, he no longer suffers from the painful intestinal illness. He's often referred to as the first child saved by next generation sequencing, and his story highlights the power of these technologies in clinical settings.
It's really amazing that PCR and next generation sequencing have actually enabled the very personalized method for treatment of disease. But are there limitations to using PCR in a clinical setting?
Absolutely, and there are several that come to mind. First, PCR often requires prior knowledge of the target.
Yeah, and so in order to amplify, for example, the cryptic plasmid from chlamydia trachomatis, the sequence of that plasmid is what needs to be known at least partially. Target information is not always available and can sometimes be difficult to obtain. Second, clinical samples of DNA or RNA often contain inhibitors, and these are substances that prohibit amplification and can generate false negative results. Proper assays controls are critical to identifying when PCR inhibition is problematic. Advances in PCR reagents, including the development of novel enzymes, have helped to minimize the effects of inhibition on amplification, but have yet to eliminate the problem completely.
Third, PCR requires a thermocycler to heat and cool the reactions. These instruments require a significant amount of power. They're not easily portable and can not be typically used in the field. Portable handheld PCR devices that operate by battery are in development, but have yet to be wildly commercialized. Finally typical PCR assays require trained laboratory personnel to execute. The holy grail of clinical diagnostic testing is the development of point of care diagnostics, which can be performed by non-skilled individuals with results obtained at the time it takes for a typical doctor's visit, roughly 30 minutes.
So say, well, I'm at the doctor's I could get tested for influenza and know before I leave the doctor's office whether or not I'm infected.
Yeah, absolutely. And while PCR has been successfully performed in as little as 15 seconds, additional work really needs to be done before it becomes mainstream as a diagnostic tool at home or even at the doctor's office.
We've spent a lot of time discussing PCR as a clinical diagnostic. Are there any other interesting uses of PCR that you'd like to tell us about?
There are so many amazing uses of PCR that we could be here all day discussing them. So given that we're short on time, I'll mention just one additional use. PCR is frequently used in the research community to stitch together pieces of DNA, ultimately modifying the genomes of various organisms in a process known as genetic engineering. Genetic engineering allows us to make proteins in the lab to better understand their roles within the cell or modify organisms to study disease.
Zebra fish, the fresh water fish that you can find at basically every pet store, is frequently used as a subject in genetic engineering experiments. Zebra fish embryos can easily be injected with DNA and they developed quickly making them a great subject for research projects. Scientists used PCR to engineer zebra fish to glow in the dark in response to pollution to better understand the impacts on animal and human health.
Yeah. Modification of zebra fish is so established that you can visit your local pet store and often find genetically engineered glowing fish available for purchase.
Fun. Thanks so much for taking the time out of your schedule to join us today. This was super interesting. It's amazing how many different ways PCR really touches our lives every day.
I'm glad you enjoyed it.
Thanks for joining us for this episode of our podcast, as always check out the transcript of this podcast for links to additional information. And be sure to tune in to our next episode featuring the “Godfather of Synthetic Biology” himself, Tom Knight. Tom will share with us the lessons he has learned by applying the problem solving techniques of computer engineering to biological quandaries.
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