PCR (Polymerase Chain Reaction) Explained

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!