Polymerase Chain Reaction (PCR) and Reverse Transcriptase-PCR (RT-PCR)
COVID-19: What You Need to Know (CME Eligible)
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Register for this free CME course here: https://www.coursera.org/learn/covid-19-what-you-need-to-know?edocomorp=public-health-free-courses COVID-19 is a global pandemic that has already resulted in hundreds of thousands of infections and thousands of deaths, with many more anticipated. This course is a go-to resource that will be regularly updated with all of the current information put forth by the CDC, WHO, and other leading agencies and covers the basics, personal protective equipment, diagnostics, and other material. As a healthcare professional, you can earn CME credit and help to #RaiseTheLine by becoming prepared to treat COVID-19.
Amazing Course..!! A very detailed information about COVID-19.\n\nCT, PCR and RT-PCR tests and science behind these tests explained in detail.\n\nThanks to the entire team for this wonderful course.
The course was very comprehensive and the illustrations and diagrams broke things down very well. It was complicated information, but I was able to take it in because of how well it was explained.
Register for this free CME course here: https://www.coursera.org/learn/covid-19-what-you-need-to-know?edocomorp=public-health-free-courses COVID-19 is a global pandemic that has already resulted in millions of infections and hundreds of thousands of deaths, with many more anticipated. This course is a go-to resource that will be regularly updated with all of the current information put forth by the CDC, WHO, and other leading agencies and covers the basics, personal protective equipment, diagnostics, and other material. Becoming familiar with the signs, learning how to manage the symptoms, and doing your part to mitigate the spread of COVID-19 will reduce the burden on our healthcare system and raise the line. This course will teach you to:- Recognize the symptoms, diagnosis, and treatment of COVID-19- Identify how COVID-19 has spread around the world- Apply into practice the public health measures we need to help mitigate the spread of COVID-19. As a healthcare professional, you can earn CME credit and help to #RaiseTheLine by becoming prepared to treat COVID-19. 681 thousand deaths
Polymerase Chain Reaction (PCR) and Reverse Transcriptase-PCR (RT-PCR)9:00
Rishi Desai, MD, MPH
Chief Medical Officer, Osmosis
Mary W. Ales, CCMEP, FACEHP
Executive Director, IPMA
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Our DNA contains loads of information, neatly stacked and compressed to insanely small sizes able to fit within a cell nucleus. A single DNA molecule has two strands, which wrap around one another to form a double helix. Each single strand of DNA is composed of a sequence of four types of nucleotides, which are the individual letters or building blocks of DNA. Nucleotides of DNA are made up of a sugar, deoxyribose, a phosphate, and one of the four nucleobases; adenine, cytosine, guanine, and thymine, or ACGT for short. The nucleotides on one strand form hydrogen bonds to complimentary nucleotides on the other strand. Specifically, A bonds with T via two hydrogen bonds, and C bonds with G via three hydrogen bonds. Additionally, the two DNA strands also have a direction, meaning one of them runs from the three prime end to the five prime end, while the other one runs from the five prime end to the three prime end. Like to snakes coiled up together, but facing in different directions. Every single protein of our body is encoded through combinations of just four nucleotides. When there are errors in our genetic information, diseases occur. Let's be honest, we're always interested in knowing what was written in our DNA. Polymerase chain reaction, or PCR for short, is a technique used in molecular biology to amplify a segment of DNA. Let's take a step back. A single copy of DNA is not very much of it. So to work with DNA, we basically have lots and lots of copies of it so that it's easier to analyze. For example, if we want to visualize it, we can use a technique called gel electrophoresis. PCR is based on DNA replication, a process that our cells normally use to duplicate their genetic material before dividing into two identical daughter cells. So first of all, we're going to need a machine called a thermal cycler, that's where the PCR magic happens. You can think of it like a cauldron filled with a solution where genetic wizards add the ingredients. The ingredients are that DNA that we wish to multiply, an enzyme called Taq polymerase, specific primers that bind to the DNA, and a mixture of free nucleotides, A, T, C, and G. Throw everything in the thermal cycler, wave your magic wand, and the process begins. So let's say that we have a long double-stranded DNA molecule, and we're interested in the highlighted part. These two strands would be the template strands. The first step of PCR is denaturation, meaning that we heat up our ingredients to exactly 96 degrees Celsius, almost as hot as boiling water. This breaks open all bonds between the two strands of DNA, so that they can separate from one another. The second step is called annealing. Here's where we need primers. For our highlighted sequence, the primers would look like this. Three-prime end, CGGATAC, five-prime end, and the five-prime end, AGGTCAC, three-prime end. During the annealing step, we cool everything down to around 55 degrees Celsius. This allows the primers to bind to their complementary sequences on the single-stranded DNA. The third step is called extension. We want to make the environment as optimal as possible for Taq polymerase to do its job. See, Taq polymerase is actually a really cool enzyme. We get it from a bacteria called Thermus aquaticus that grows in geothermal hot springs. So not only can Taq polymerase withstand the heat during the denaturation step of PCR, but it also functions best at around 72 degrees Celsius. So during extension, we heat everything back up to 72 degrees, and Taq polymerase latches onto the primers, grabs some free nucleotides, and assembles the new DNA strands. That's pretty much all there is to it. A whole PCR cycle lasts roughly 10 minutes. At the end, we've doubled the amount of DNA because each template strand of DNA is now double-stranded. So now, we just continue cycling these three steps a couple of dozen times after every step, and the quantity of DNA gets doubled each cycle. Two strands, then four strands, then eight, then 16, then 32, then 64, then 128, and so on and so forth. So PCR actually multiplies DNA at an exponential rate. After around six hours and 40 cycles, we'll theoretically have two to the power of 40, or 1,099,511,627,776 copies. But that's easier to remember as just a whole lot of DNA that we can analyze further. There are a lot of variations of PCR, but let's discuss one that has been used recently for diagnosing of viral infection. Specifically, what if you wanted to diagnose someone with COVID-19? To do that, you would collect a nasal pharyngeal swab, which is where you take a long Q-tip with only one soft end, and twirl it a few times inside a nostril to get enough secretions on it to be analyzed. Now, getting a good sample is crucial, so make sure you really get in there. If no virus gets on the swab, then PCR can't detect it. To diagnose COVID-19, what you would look for is a virus called severe acute respiratory syndrome, Coronavirus 2, or SARS-CoV-2. Now, SARS-CoV-2 is an RNA virus, not DNA. So to detect, you would need to use a particular type of PCR called reverse transcriptase PCR or RT-PCR for short. This technique is little different because it starts with RNA instead of DNA. If you remember, one difference between RNA and DNA is that RNA doesn't have thymine, instead it has uracil. RNA is complementary to DNA though, so if an RNA strand has a nucleotide sequence three-prime, AAG UCC AGU, five-prime, then the complementary DNA or cDNA strand would be five-prime, TTC AGG TCA, three-prime. To perform RT-PCR, some of the extracted sample from the nasal pharyngeal swab is added to a solution containing an enzyme called reverse transcriptase, nucleotides and primers that are complimentary to a specific SARS- CoV-2 target sequence. If the viral RNA is present, the primer is attached to the RNA strand and then reverse transcriptase synthesizes a cDNA strand. Once we've got the cDNA, the steps of RT-PCR are pretty much the same as PCR. Taq polymerase is used to amplify the cDNA through denaturation, annealing, and extension steps. After several rounds, if there was SARS-CoV-2 RNA in the original sample, you'll have amplified DNA from that sample that can be detected. As a quick recap, polymerase chain reaction is a technique used to massively multiply DNA so that it can be analyzed using other techniques. It requires a thermal cycler filled with the DNA sample to be multiplied, thermal stable Taq polymerase, primers with which we select what gets multiplied, and free nucleotides of all four types, A, T, C, and G. The first step is denaturation, then there's annealing, and finally extension. PCR doubles the amount of DNA that we're interested in looking at in each cycle.