Polymerase Chain Reaction (PCR) Test Explained

The polymerase chain reaction, more commonly referred to as PCR, is sometimes explained as “molecular photocopying.” PCR is an inexpensive and convenient way to copy or “amplify” small chains of DNA into samples larger enough for molecular and genetic analyses.

PCR uses relatively short DNA sequences known as primers to choose the portion of the genome to be amplified. The sample is buffered in a saline solution to keep pH constant, and DNA-replicating enzymes are added. The temperature of the mixture is raised and lowered repeatedly over a period of several hours to assist a DNA replication enzyme in copying the primer. The result is up to a billion copies of the prime DNA sequence in just a few hours.

Once a strand of DNA has been amplified by PCR, the product can be used in a variety of laboratory procedures. PCR is a first step in testing for many kinds of bacteria and viruses, including HIV and COVID-19. Amplified DNA is useful in the detection of many genetic disorders. And the human genome project would not have been possible without PCR.

Prior attempts to decode the human genome had relied on techniques that would begin at one telomere of a strand of DNA and attempt to read millions of bases until it reached the telomere at the other end. With PCR, however, the human genome project sequenced multiple strands of DNA at different places but at the same time. The cost of reading the human genome was reduced by a factor of over 100,000 and sped up by years.

There are a huge number of applications of PCR beyond the human genome project. PCR might be used by a researcher who wants to study a single gene. Or maybe a crime scene detective is looking for a genetic marker to rule out a suspect or justify further investigation. Or an ancestry service may want to match DNA sequences between people who don’t know each other to confirm that they are biologically related.

PCR is widely regarded as one of the most important advances in the study of molecular biology in the twentieth century. Its creator, Kary B. Mullis, was awarded the Nobel Prize for Chemistry in 1993.

PCR Process Steps Explained

The three PCR steps seem almost intuitive since the technique has been so widely used, although the invention of PCR was revolutionary in its time. Before the steps make sense, however, there are two inputs into PCR testing about which we need to give further detail.

Taq Polymerase

Cells have to replicate their DNA with a DNA polymerase before they can divide. The DNA polymerases that power mitosis make new strands of DNA on existing strands of DNA they use as templates.

PCR uses a specialized polymerase known as the Taq polymerase. This polymerase gets its name from the thermophilic bacterium in which it was first isolated, Thermus aquaticus. This bacterium lives in thermal vents of underwater volcanoes. It survives at temperatures that would denature proteins and enzymes in most other species. Its polymerase is optimally active at temperatures around 70° C, or 158° F for your students.

Heat stability is what makes the Taq polymerase the ideal choice for PCR. This procedure makes repeated use of high temperatures to denature and separate the strands of DNA as they are replicated.

PCR Primers

Polymerases, including Taq polymerase, can only make DNA if they are given a template, the primer we mentioned previously. Every polymerase uses a short sequence of A-C-G-T nucleotides as a starting point. The experimenter chooses the primers that will be amplified by the Taq polymerase in PCR.

Primers are very short chains of DNA, usually around just 20 nucleotides long. Every PCR reaction uses two primers chosen to flank the target region to be copied. This way the primer bind to opposite strands of DNA, at both edges of the segment the experimenter seeks to copy. The primers then bind to the DNA template by pairing complementary bases. An A-G-C-T-A-G-C-T-A-G-C-T-A-G-C-T template would bond to a T-C-G-A-T-C-G-A-T-C-G-A-T-C-G-A primer.

Once the primers bind to the template, they are extended by the Taq polymerase, and the nucleotides between them will be copied.

Those basics out of the way, we can now talk about the 3 steps of PCR.

3 Steps of PCR

The essentials of PCR are the Taq polymerase, template DA, primers, and the A-G-C-T nucleotides. The experimenter assembles these ingredients in a tube, along with the cofactors that regulate the kinetics of the Taq enzyme, and puts the mixture through repeated heating and cooling that power the synthesis of DNA.
The three basic steps are:

Denaturation at 96° C, heating the polymerase, DNA to be copied, primers, and nucleotides so strongly that the sample DNA is denatured. Its strands unravel. Denaturation converts double-stranded DNA to single-stranded DNA for the next step.
Annealing at 55 to 65° C cools the reaction, so the priers can bind to their complementary nucleotide sequences
Extension at 72° C raises the reaction temperature so Taq is active, extending the primers, and synthesizing new strands of DNA.

PCR repeats this cycle 25 to 35 times over a typical reaction that takes two to four hours. Copying shorter segments of DNA takes less time, and copying longer segments of DNA takes more time. Efficient PCR reactions can create billions of copies of DNA, because it’s not just the original DNA that is used as a template each time. Every copy of DNA serves as a template for the next round of synthesis.

The number of DNA molecules roughly doubles with each reaction. As a result, 25 rounds of PCR can create 2 25 or over 30 million copies of the DNA being studied, and 35 rounds of PCR can create 235 or over 34 billion copies of the target DNA.

Visualizing PCR Results with Gel Electrophoresis

The next step in characterizing DNA (usually) is visualizing the results of PCR with gel electrophoresis. As students of teachers who use Modern Biology’s gel electrophoresis supplies and all-in-one gel electrophoresis laboratory teaching kits are well aware, gel electrophoresis is a technique that uses electrostatic charge to pull segments of DNA through a gel matrix. DNA is dyed with ethidium bromide, so it glows under UV light. Experimental and control samples of DNA are placed at one end of a buffered gel (buffered with saline to keep pH stable) in wells covered by the conductive gel.

DNA has a slight negative charge, so DNA samples are placed nearest the cathode, the negative charge attached to the gel. They are pulled toward the anode, the positive charge at the other side of the gel. Shorter segments get pulled faster than longer segments, so when DNA is visible at the anode side of the gel, power is turned off.

If everything has gone as planned, the electrostatic charge has created a “ladder” of DNA samples with known base pair lengths at one side of the gel, and the tested DNA has arrived near or between two controls that give a rough estimate of how many base pairs are in the sample. The length of the DNA in the sample is now estimated, and the millions or billions of copies of test DNA now visible under UV light, can be cut out of the gel for further analysis.

Practical Applications of PCR

One of the questions that comes up in biology classes at all levels since the beginning of the pandemic is “What is a COVID-19 test, anyway?” The answer is, although there are several approaches to COVID-19 detection, the most commonly used method is a highly automated polymerase chain reaction test.

Real-time PCR amplifies a known primer of COVID-19 DNA that does not carry the risk of causing disease. Amplifying this segment of DNA tens of millions of times, that is, if it is to be found on the swab, automated detection equipment can detect COVID-19 with 97-percent specificity and 100-percent sensitivity. Chances are that many of your students will have had PCR testing.

PCR, of course, has many other applications that pique student interest. PCR can be used to amplify fetal DNA from a tiny sample of amniotic fluid to test for genetic issues. PCR can amplify bacterial or viral DNA to test for the presence of pathogens in a patient’s bone sample, skin sample, serum, or spinal fluid.

PCR is of enormous value in forensics. The DNA in a single hair follicle can be amplified for testing that can exclude an innocent suspect or point to the need for further investigation to establish probable cause for an arrest.

Modern Biology provides everything busy biology instructors need to introduce their students to hands-on experiences of PCR. Our experiments aren’t just demonstrations of PCR. Your students will use PCR to confirm or disconfirm a scientific hypothesis of their own making. And if you want to go beyond our experiments, we have all the PCR supplies you need.

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