Welcome back to BOGObiology! In this video we'll be discussing Polymerase Chain Reaction or "PCR". We'll be learning about what PCR is, how it's used, its reagents the steps in the PCR process, and how PCR is used in covid testing.
PCR or "polymerase chain reaction" is a genetic copying process used in biotechnology. Biotechnology is a rapidly growing field that harnesses naturally occurring processes for useful purposes. In the case of PCR, it harnesses the process of DNA replication to make copies of genetic material.
PCR is sometimes referred to by the nickname "molecular photocopying". PCR makes millions to billions of copies from a small amount of genetic material. The number of copies doubles with each cycle of the reaction, resulting in exponential growth.
PCR is an easy way to replicate a small sample so there is enough to study, analyze, or use in other reactions. PCR is used in many industries but the most familiar are likely forensics, agriculture and medicine. In forensics, PCR can be used to amplify genetic material from an unknown source so that it can be compared to a particular suspect or to a large DNA database.
DNA evidence is now routinely used to solve crimes. In agriculture, PCR is a key part of plant genotyping for breeding so that desirable traits can be combined, or to determine if a particular plant should be cloned. In medicine, PCR is a diagnostic tool used in genetic testing, tracking cancer mutations, and of course in covid testing.
If you're aiming to perform PCR, several reagents must be combined: A DNA sample, a polymerase enzyme, deoxynucleosides, primers, buffers and cofactors. The DNA sample contains the sequence of interest to be copied. This will become known as the template strand.
The enzyme DNA polymerase will be used to build the new DNA strands. Most types of DNA polymerase would be denatured by the hot temperatures used in the PCR reactions. So for these purposes, we use a special polymerase enzyme called Taq Polymerase.
This enzyme has been isolated from the heat resistant bacteria thermus aquaticus, and can withstand the hot temperatures needed for PCR. Deoxynucleoside triphosphatases or DNTPs are the building blocks used to construct the new DNA copies. Sometimes these are also called free nucleotides.
The Taq polymerase will arrange the DNTPs using the template strand as a guide. Primers are single stranded chunks of DNA that are complementary to the start of a target region. They tell the Taq polymerase exactly where to start copying.
Because it's so important for the primers to mark only the correct part of the template strand, a tremendous amount of effort goes into designing them. We need to add two additional reagents to give them the best possible chance of working properly. The PCR process also needs two more reagents to create the ideal conditions for amplification: a buffer solution and a magnesium cofactor.
Buffer solutions help to maintain optimal conditions for a reaction to occur. Often this means maintaining the pH, or concentrations of particular ions. There are many types of buffers and they're common in biotech.
The magnesium cofactor has two roles; it helps the primers to adhere at the correct site and it helps the Taq polymerase enzyme to function optimally. Usually these magnesium ions are added to the mixture in the form of MgCl2. PCR mimics many aspects of the natural process of DNA replication.
DNA is a double-stranded molecule and it needs to be replicated faithfully before the cells can divide. In nature, one enzyme separates the two strands and then another creates a complement to each strand resulting in two identical copies. PCR is a very similar process but it uses a machine called a thermocycler rather than a cell.
The steps of PCR are denaturation, annealing, and extension, and the machine uses heat to control the reaction. During the denaturation phase, we heat the DNA to approximately 95 degrees celsius (which is almost boiling if you're someone who is used to fahrenheit! ) This breaks the hydrogen bonds in the middle of the molecule and creates two template strands for copying.
The second phase of PCR is annealing which takes place at about 55 degrees celsius; much cooler than the denaturation phase. During the annealing phase, primers flank the sequence of interest on the template molecule in preparation for copying. Primers are single-stranded DNA fragments that are designed to stick to a very specific part of the sample.
It's very important that the primers stick to the correct spot of the DNA sample because that's the area that's going to be copied. Primers are usually 18 to 22 bases in length with a maximum of about 30. However, finding the correct length is a balancing act; shorter primers easily stick to the template strand but they are also much more prone to annealing in the wrong place.
A primer's tendency to stick only in the correct place is called specificity. The annealing stage is where the magnesium first comes into play. The template strand has a negatively charged backbone and the primers have a negatively charged backbone.
These would usually repel each other, preventing the primer from sticking. The positively charged magnesium ions keep the primer in the correct orientation so it can more easily bind to the correct site. The buffer also helps with the primer annealing process; if the primer sticks correctly the buffer will stabilize the hydrogen bonds that form between the template and the primer.
If the primer happens to adhere in the wrong place, the buffer will destabilize the bond. The third stage is extension which occurs at about 72 degrees celsius. This is where the "polymerase" of polymerase chain reaction finally comes onto the scene.
The Taq polymerase enzyme moves along the template strand from the primer starting point in the five prime to three prime direction. Remember that polymerase can move only in this direction. The Taq polymerase creates a new complementary strand out of the DNTPs that we added to the reaction.
At the end of the extension phase, the amount of DNA has doubled. Magnesium also plays a role in the extension phase. It helps to form a bond between the three prime end of the primer and the phosphate group of the first DNTP.
PCR is a cycle; the denaturation, annealing, and extension phases repeat over and over, doubling the number of copies each time. After one cycle, there are 2 copies, then 4, then 8, then 16, etc. After 25 cycles there will be 2 to the 25 or roughly 33 million copies.
Even though the original sample may have been quite small, if the process has been performed correctly there will now be plenty of material for analysis. PCR has long been used in diagnostics but with the rise of the Covid 19 global pandemic, PCR testing has become far more widely known. PCR testing for the virus uses a modified version of PCR called RTq-PCR also known as "reverse transcription quantitative pcr".
The name RT-qPCR sounds like a confusing jumble of random letters, so we're going to break it down step by step beginning with the "RT" portion. Many viruses including SARS-CoV-2 the virus that causes covid 19, use single stranded RNA rather than DNA as their genetic material. To do PCR on it, we need to add some additional reagents to the PCR mixture.
Reverse transcriptase is a naturally occurring enzyme that can be used to manufacture DNA by using RNA as the template. This is the reverse of the usual DNA to RNA process. We call this copy cDNA, short for "complementary DNA".
The primers are made specifically to only adhere to the viral RNA; only the viral genetic material if it's present will be duplicated. Once the cDNA is created from the RNA it can be copied using PCR just like regular DNA. We can't tell whether the viral genetic material is present in the tube just by looking at it, so this is where the "q" or "quantitative" part of RT-qPCR becomes important.
Like standard PCR, RT-qPCR makes copies of a specific region of interest but it also measures the amount of genetic material in a sample by using fluorescence. Adding a glowing reagent into the DNA allows a machine to measure or quantify how much DNA is in a sample. As the amount of genetic material grows exponentially, a computer measures and records the level of fluorescence on a chart.
The amount of material doubles with each cycle; if the patient's sample contained viral RNA, the tube will grow brighter and brighter as the process continues. If the sample contained no viral RNA, the tube will remain dark. There are two sets of fluorescent reagents that are commonly used (as of the making of this video) SYBR Green and Taqman probe.
Both reagents have been shown to be equally accurate in detecting the presence of SARS CoV-2. SYBR Green is a dye that attaches to double-stranded DNA but will not attach to single-stranded DNA. As the amount of DNA doubles, the amount of fluorescence will increase as well.
In a Taqman probe assay, short sequences called probes are added to the reaction mixture. They're built to temporarily attach to a specific target sequence, in this case a particular part of the viral DNA. The probe contains a glowing reporter component along with something called a quencher that keeps the reporter turned off as long as the two molecules are close together.
As the Taq polymerase builds the new complementary strand, it will dislodge the probe and break it into pieces. With the reporter and the quencher separated, the reporter will start to glow. As the number of broken probes grows exponentially as the reactions progress, the sample will grow brighter and brighter.
If the level of fluorescence surpasses a certain threshold, the patient is considered to have tested positive. Because the sample will only start to glow if viral genetic material is present, false positives are very rare. Without viral RNA in the sample, the primers will not attach, the reaction will not proceed, and you won't get any glowing.
False negatives, however, do sometimes occur. For instance if a patient is tested quite early after being infected there may not be enough viral genetic material in the patient's sample for it to be detectable. That wraps up our discussion of PCR!
