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The polymerase chain reaction (PCR) is a rapid, specific and sensitive in vitro enzymatic method of amplifying specific DNA sequences. This process was conceived by Kary Mullis in 1983.
The yield from the procedure is able to provide enough copies for probe detection or identification. This technology is highly sensitive: PCR can detect and amplify as little as one DNA molecule in almost any kind of sample.
Specificity is based on the use of two oligonucleotide primers that hybridize to complementary sequences on opposite strands of DNA and flank the target sequence.
Reverse transcription polymerase chain reaction is currently used as a detection tool for COVID-19.
Read more about coronavirus infectious disease 2019 and its infection cycle here.
Terms definition
In the quest to understand polymerase chain reaction it is important to understand the meaning of the following terms;
- Reverse genetics –This is the generation of an RNA virus genome from a cloned copy DNA.
- Reverse transcriptase This is an enzyme that can synthesize DNA using (a) an RNA template and (b) a DNA template
- Reverse transcription is the synthesis of DNA from an RNA template
- Reverse transcription polymerase chain reaction (RT-PCR) is an in vitro technique for amplifying the data in RNA sequences by first copying the RNA to DNA using a reverse transcriptase. The DNA is then amplified by a PCR.
Polymerase chain reaction concept
The simple concept of the PCR relies upon the repeated synthesis of the targeted DNA by DNA polymerase enzyme.
There are three steps needed to achieve this synthesis, that are carried out repeatedly and in succession at different and controlled temperatures, allowing for the doubling of the amount of DNA after each cycle.
Reverse transcription polymerase chain reaction is a variation of polymerase chain reaction that is used to measure RNA expression levels. In this technique the complementary DNA is made by use of an enzyme known as reverse transcriptase that transcribes the mRNA templates.
DNA molecule contains a lot of information stacked together
A single DNA molecule is made of two strands wound around to form a double helix structure. A single strand is made up of building blocks known as nucleotides. These nucleotides are labeled using letters
A nucleotide is made up of a neucleobase, sugar known as deoxyribose and a phosphate .
The neucleobases in a nucleotide are thymine, adenine, guanine and cytosine.
These nucleotides form hydrogen bonds to connect with complementary nucleotides on another strand.
When a sample is likely to contain a low number of copies of a virus nucleic acid the probability of detection is increased by amplifying virus DNA using a polymerase chain reaction technique, while RNA can be copied to DNA and amplified using a RT (reverse transcriptase)-PCR.
The procedures require oligonucleotide primers specific to viral sequences.
An amplified product can be detected by Southern blotting, or direct sequence determination or gel electrophoresis in an agarose gel, followed by transfer to a nitrocellulose membrane, which is incubated with a labelled probe.
There are also PCR techniques available for determining the number of copies of a specific nucleic acid in a sample. Real-time PCR is commonly used for this purpose.
In Real time PCR technique, the increase in DNA concentration during the PCR is monitored using fluorescent labels; the larger the initial copy number of DNA, the sooner a significant increase in fluorescence is observed.
The PCR cycle at which the fluorescent signal passes a defined threshold is determined and this gives an estimate of the starting copy number
Steps in PCR
These three steps are:
- Denaturation. Denaturation process requires heating of the specimen to a high incubation temperature of about 90-98°C. At this temperature the bonds holding the double-stranded DNA containing the target region melt and form separate single strands.
- Annealing. Annealing involves lowering of the temperature of the denatured single strands typically to 37-65°) in order to allow two synthetic oligodeoxyribonucletides known as primers to anneal to sites flanking the region of DNA to be amplified. One for each strand.
The four deoxynucleoside triphosphates are then added, and the primed DNA segment is replicated selectively.
Primers are added to the reaction mixture in a molar excess so as to favour formation of the primer-target complex instead of renaturation of the target DNA.
At the end of annealing process, the primers are annealed to opposite strands of the DNA with their 3’ ends facing each other.
An automatic change in temperature is effected using a machine known as a thermocycler. - Extension. This step entails the alteration of the temperature to the optimum so as to allow the DNA polymerase to abstract deoxynucleotides (dNTPs) from the reaction mixture for the 5’ to 3’ synthesis of DNA directed from the primers, using the target DNA as template.
This means that, the DNA polymerase adds nucleotides to the 3'-hydroxyl end of the primer, and strand growth extends across the target DNA, making complementary copies of the target.
Through this process the primers become incorporated into the PCR product.
The two DNA strands each serve as a template for the synthesis of new DNA from the two primers.
Typically 20–30 cycles are run during this process, amplifying the DNA by a million-fold (220) to a billion-fold (230). Each extension product includes a sequence at its 5'-end that is complementary to the primer.
Thus, each newly synthesized strand can act as a template for the successive cycles. This leads to an exponential increase in the amount of target DNA with each cycle hence the name “polymerase chain reaction.”
The use of a DNA polymerase enzyme for thymus aquaticus, an extreme thermophilic bacterium, known as Taq DNA polymerase makes the execution of the polymerase chain reaction more amenable to automatic. Since this enzyme is unaffected by temperature fluctuation, there is no need for it to be added after each cycle.
One cycle of PCR produces double target sequence and flanked by primers. These extension products are simply complementary to the main DNA for analysis and can bind to primers for further cycles.
Reverse Transcriptases have the potential to copy any RNA into DNA, even in the absence of specific transfer RNA primers. The two most commonly used reverse transcriptases are those from avian myeloblastosis virus and Moloney murine leukaemia virus.
When comparing the mRNA expression level of a certain gene using realtime polymerase chain reaction (RT-PCR), the target gene is amplified in real-time PCR and the expression level is calculated using the cycle threshold (Ct) values.
For accuracy, the RNA must be extracted from the same number of cells, and amplification of the target RNA must be conducted at the same level of efficiency.
since amplification products double in number every cycle, the difference in Ct value implies exponentiation of two, but PCR does not follow this theory. Therefore, the amplification efficiency should be compared for accurate quantification, even if the products are amplified using the same primers.
The cycle reflecting this amplification efficiency is an analytical curve. The analytical curves are amplified with a series of samples of differing concentration, making a graph of the concentrations of the samples, and obtaining the relative Ct values. When measuring the relative amounts of samples, they can be compared on the analytical curve.
Principles of reverse transcriptase polymerase chain reaction
Reverse transcriptase polymerase chain reaction has two steps
- Synthesis of complementary DNA known as cDNA,
- Conventional polymerase chain reaction using primers.
In RT-PCR the measurement of specific RNA is achieved by monitoring amplification reaction with fluorescence.
RT-PCR can be one step or two step.
The difference between the two approaches is the number of tubes used when performing the procedure.
One step RT-PCR
This means that the entire reaction from the reverse transcription to form cDNA synthesis to polymerase chain reaction amplification occurs in a single tube. This minimizes experimental variation by containing all of the enzymatic reactions in a single environment.
This process is less accurate compared to the two-step approach.
Two step TR-PCR
In the two-step reaction, the reverse transcriptase reaction and PCR amplification are performed in two separate tubes.
Quantification of RT-PCR products can largely be divided into two categories: end-point and realtime PCR.
End-point RT-PCR
The measurement approaches of end-point RT-PCR requires the detection of gene expression levels by the use of fluorescent dyes such as ethidium bromide, P32 labeling of PCR products using phosphorimager,or by scintillation counting.
End-point RT-PCR uses three different methods: relative, comparative and competitive.
Real-time RT-PCR
Real-time RT-PCR is the method of choice for quantification of gene expression; it is also the preferred method of obtaining results from array analyses and gene expressions on a global scale.
It uses four different fluorescent DNA probes for the detection of PCR products:
- TaqMan probe,
- Molecular Beacons probe,
- SYBR Green probe, and
- Scorpions probe.
All of these probes allow the detection of PCR products by generating a fluorescent signal.
Requirements
For one to perform a polymerase chain reaction they require;
- A thermal cycler is filled with DNA sample to be multiplied,
- A thermostable Taq polymerase enzyme,
- Primers with which we select what gets multiplied (The oligonucleotides serve as replication primers that can be extended by DNA polymerase)
- Free nucleotides.
Reverse transcribing viruses
These viruses replicate using reverse transcription, which is the formation of DNA from an RNA template.
Reverse transcribing viruses containing RNA genomes use a DNA intermediate to replicate, whereas those containing DNA genomes use an RNA intermediate during genome replication. Both types use the reverse transcriptase enzyme to carry out the nucleic acid conversion.
Retroviruses often integrate the DNA produced by reverse transcription into the host genome. Retroviruses belong to a family of Retroviridae. They are enveloped, single stranded positive sense RNA viruses that replicate through a DNA intermediate using reverse transcriptase.
This large and diverse family includes members that are oncogenic, are associated with a variety of immune system disorders, and cause degenerative and neurological syndromes. Reverse transcriptase polymerase chain reaction is important in detecting the genetic material of these viruses.
Uses of RT-PCT
This technique is used in;
- Diagnosis of genetic diseases such as Lesch-Nylan Syndrome by detecting the defective HPRT1 gene.
- Besides screening for genetic mutations, PCR can be used to detect and identify infectious agents such as Trypanosoma cruzi, the causative agent of Chagas’ disease, and Neisseria meningitides, the causative agent of bacterial meningitis, through the selective amplification of their DNA.
- Making prenatal genetic diagnoses,
- Detection of allelic polymorphisms
- Measurement of gene expression
- Gene insertion,
- Establishment of precise tissue types for transplants;
- Cancer detection. This is made possible because circulating cancerous tumors produce a unique messenger RNA transcript depending on the type of the cancer.
- Study of genomes of RNA viruses such as retroviruses, influenza virus and coronaviruses.
- Scoring in vivo protein–DNA occupancy using chromatin immunoprecipitation assays
Challenges
Exponential growth curve of the reverse transcribed cDNA produces inaccurate end product quantification due to the failure in maintaining linearity.
A simple slight DNA contamination can cause undesirable results. These false positive or negative results are eliminated by use of tags or anchors to the 5’ region of the gene specific primer.
