What is polymerase chain reaction

Polymerase chain reaction or PCR sometimes called “molecular photocopying,” is a laboratory procedure in which millions to billions of copies (amplicons) of a specific piece of DNA are made. Polymerase chain reaction is essentially an amplification method, whereby the tiniest amounts of DNA that may be present in blood, hair or tissues can be copied so that there is enough for analysis. The polymerase chain reaction (PCR) is a fast and inexpensive technique used to “amplify” – copy – small segments of DNA. Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without polymerase chain reaction amplification.

The name of polymerase chain reaction is derived from the key component involved that carries out the replication of the DNA, called a DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. DNA polymerase is an enzyme that exists in nature. The most commonly used DNA polymerase is Taq polymerase, which is obtained from the bacterium Thermus aquaticus. The Taq polymerase enzyme works optimally at about 158 °F (70 °C). It can create a new DNA strand, using the original DNA as a template, and using DNA oligonucleotides (also known as primers). The primers used in polymerase chain reaction are synthesized, short sequences of DNA that are made to match exactly the ends of the DNA region to be copied.

Polymerase chain reaction has replaced previous methods of DNA replication that used bacteria and could take several weeks to complete. Polymerase chain reaction can be done within a few hours, making it a very rapid assay. Speed is often required in a diagnostic setting when urgent results are necessary.

Polymerase chain reaction was developed around 1983 by Kary Mullis, who won a Nobel Prize in Chemistry for the invention in 1993. Since then, polymerase chain reaction has been widely used as a diagnostic and research tool. Its applications are continually growing and are widespread over many scientific disciplines, including molecular biology, microbiology, genetics, clinical diagnostics, forensic science, environmental science, hereditary studies and paternity testing.

What is the purpose of the polymerase chain reaction

Once amplified, the DNA produced by polymerase chain reaction can be used in many different laboratory procedures. For example, most mapping techniques in the Human Genome Project relied on polymerase chain reaction.

Polymerase chain reaction is also valuable in a number of laboratory and clinical techniques, including DNA fingerprinting, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders.

Detecting infectious agents

Polymerase chain reaction is extensively used in analysing clinical specimens for the presence of infectious agents, including HIV, hepatitis, human papillomavirus (the causative agent of genital warts and cervical cancer), Epstein-Barr virus (glandular fever), malaria and anthrax.

polymerase chain reaction is particularly invaluable in the early detection of HIV as it can identify the DNA of the virus within human cells immediately following infection, as opposed to the antibodies that are produced weeks or months after infection. polymerase chain reaction can also be used to determine the viral load (i.e. how much virus is circulating around the body), which is a useful measure of prognosis.

Malaria is traditionally diagnosed by identifying malarial parasites (Plasmodium falcipurum) through microscopic analysis of the blood. However, polymerase chain reaction technology has been useful in that it can rapidly identify the species of malaria present. This is important in cases of mixed infection, and also in determining the type of drug treatment to use. Currenty, polymerase chain reaction is used to complement microscopic examination.

polymerase chain reaction can be used to identify the bacterium Bacillus anthracis, the causative agent of anthrax. Because of the need to rapidly diagnose such infections, polymerase chain reaction has become an important tool in detecting the presence of anthrax in clinical specimens. It replaces conventional methods of using a specimen to grow the bacteria in the laboratory, which take at least 24 hours. polymerase chain reaction provides a rapid, sensitive and specific alternative.

Polymerase chain reaction in cancer diagnostics

Polymerase chain reaction is an invaluable tool as it can provide information on a patient’s prognosis, and predict response or resistance to therapy. Many cancers are characterised by small mutations in certain genes, and this is what polymerase chain reaction is employed to identify.

For example, in acute myeloid leukaemia (AML) the presence of a mutation known as t(8:21) can indicate a good prognosis, as certain drugs are known to be successful in patients who carry this mutation. The presence of a mutation in a gene called Flt3 can identify those patients who are less likely to fully respond to treatment with chemotherapy, and who have a high risk of relapsing following treatment.

Polymerase chain reaction can also be applied in monitoring leukemia patients following treatment, by counting the number of cancerous cells that are still circulating in their bodies.

Genetic diseases and paternity testing

Another important application of polymerase chain reaction is in the analysis of mutations that occur in many genetic diseases (e.g. cystic fibrosis, sickle cell anaemia, phenylketonuria, muscular dystrophy). Because of the sensitivity of polymerase chain reaction, this can be done from a single cell taken from an embryo before birth.

The paternity test is essentially carried out by polymerase chain reaction. A cheek swab is taken from inside the mouth of both parents and the child. The DNA is extracted from the cells obtained and is analysed by polymerase chain reaction. Everyone’s DNA is the same in every cell in the body. A child’s DNA should have part of the mother’s and father’s DNA. Several locations called ‘loci’ on the child’s DNA are examined, and the sequences of these loci are compared to the mother and father to see if there are matches from both parents.

How does polymerase chain reaction work?

There are three basic steps involved in performing a polymerase chain reaction. The steps are repeated 30-40 times in cycles of heating and cooling, with each step taking place at a different temperature.

Components required to carry out a polymerase chain reaction:

• A DNA template: The DNA to be copied, usually extracted and purified from blood or other tissue.
• Primers: Single stranded oligonucleotides that match exactly the beginning and end of the DNA template. These are generated synthetically.
• A DNA polymerase (e.g. Taq polymerase): To synthesise the DNA.
• dNTPs (Deoxyribonucleotide triphosphates): The building blocks from which the Taq polymerase can synthesise new DNA. These are added in excess amount.
• A buffer solution: Creates an optimal environment for the reaction to occur in.
• Magnesium chloride salt solution

To amplify a segment of DNA using polymerase chain reaction, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called “Taq polymerase” synthesizes – builds – two new strands of DNA, using the original strands as templates. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and so on. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment.

The entire cycling process of polymerase chain reaction is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis.

Polymerase chain reaction steps

All of the components are mixed together in one tube in very tiny volumes. The reaction is carried out in an automated machine, known as a thermocycler, which is capable of rapidly increasing and decreasing the temperature.

• The first step is known as the denaturation step and is carried out at around 203 °F (95 °C).

DNA exists in nature as a double stranded molecule linked together by weak hydrogen bonds. To be able to copy it, the DNA needs to be separated into single strands (denatured). This can be done by heating it to over 194 ­°F (90 °C).

• The second step is the annealing step and is carried out at about 131-140 °F (55-60 °C).

Although the temperature is lowered, an excess amount of primers prevents the denatured DNA from reforming the double helix. The primers attach or ‘anneal’ to their matching sequence on the original DNA strand.

• The final extension step is carried out at 161.6 °F (72 °C).

Taq DNA polymerase binds to the annealed primer. Taq polymerase works its way along the DNA, adding complementary nucleotides using the dNTPs and other components in the reaction mix. This completes the replication process.

• Once synthesis has been completed, the whole mixture is heated again to 203 °F (95 °C) to melt the newly formed DNA complexes. This results in twice the amount of template available for the next round of replication. Repeated heating and cooling quickly amplifies the DNA segment of interest. Roughly one million copies are made after 20 cycles.

Variations of polymerase chain reaction

There are several variations of the polymerase chain reaction technique. One commonly used and important variation is real-time polymerase chain reaction or quantitative polymerase chain reaction (q-polymerase chain reaction). Real-time polymerase chain reaction can be used to count the amount of DNA, or number of copies of a gene, that is present in a sample.

It is employed to determine the viral load of HIV in AIDS patients, and also in cancer diagnostics to count the number of cancerous cells remaining in a patient undergoing treatment.

Polymerase chain reaction limitations

As with many diagnostic tests in the laboratory, the possibility of false positive and false negative results does exist when polymerase chain reaction is used for detecting infectious agents. Therefore, follow up confirmation tests are always carried out.

One disadvantage of polymerase chain reaction technology is that it is extremely sensitive. Trace amounts of RNA or DNA contamination in the sample can produce extremely misleading results ¹. Another disadvantage is that the primers designed for polymerase chain reaction requires sequence data, and therefore can only be used to identify the presence or absence of a known pathogen or gene. Another limitation is that sometimes the primers used for polymerase chain reaction can anneal non-specifically to sequences that are similar, but not identical, to the target gene ².

Another potential issue of using polymerase chain reaction is the possibility of primer dimer formation. Primer dimer is a potential by-product and consists of primer molecules that have hybridized to each other due to the strings of complementary bases in the primers. The DNA polymerase amplifies the primer dimer, leading to competition for polymerase chain reaction reagents that could be used to amplify the target sequences ³.

References

1. Ghannam MG, Varacallo M. Biochemistry, Polymerase Chain Reaction (PCR) [Updated 2018 Dec 10]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK535453
2. Smith CJ, Osborn AM. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol. Ecol. 2009 Jan;67(1):6-20.
3. Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, Newton C, Little S. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 1997 Aug 15;25(16):3235-41