Quantitative/Real-time PCR (Polymerase Chain Reaction)

PCR (Polymerase Chain Reaction):

• The two strands of the target DNA molecule are denatured and separated by heating (94 degrees Celsius), in the first step of PCR.
• In the second step of PCR, annealing, unique sequences around 20 bp (oligonucleotides) complementary to 5′ and 3′ part of the target DNA called primers will bind. The primers are designed such that the free 3′ end of the primer faces each other. Temperature between 45 to 60 degrees celsius is chosen for this step. This temperature depends on the length and sequence of the primer.
• After the primers bind to the complementary strand of the target DNA, it is extended by DNA polymerase using four deoxynucleotide triphosphate. This is the third step. Here, the breakthrough for DNA polymerase enzyme that could be used for PCR came with the introduction of Taq polymerase. Taq DNA polymerase was extracted from the thermophilic bacteria, Thermus aquaticus. Hence, Taq polymerase is resistant to high temperatures such as the denaturation temperature. This enzyme has an optimum temperature for DNA replication at 72 degrees celsius. The heating and cooling cycles during PCR can be automated using specially designed heating blocks.

These three steps of denaturation, annealing, and extension are repeated in a cyclic manner (20-30 times) in order to amplify the DNA in between the two primer binding sites.

Quantitative PCR (qPCR)/Real-Time PCR:

Real-time PCR relies on the detection and quantitation of amplified DNA in real-time using a fluorescent reporter. The signal intensity emitted by the fluorescent reporter increases in direct proportion to the amount of the PCR product obtained.

• SYBR Green: The common fluorescent reporter used is SYBR Green where the reporter is double-strand DNA specific. This specificity allows it to detect the newly formed PCR product by binding to the minor groove of the double-stranded DNA and upon excitation, emits light. Hence, the fluorescence increases as the PCR product accumulates. SYBR green is advantageous as it is inexpensive, sensitive, and easy to use. With enough target DNA and well-designed primers, the SYBR Green is extremely efficient with non-specific background occurring in very late cycles.
• TaqMan: This is an alternative method for quantifying PCR products. This method relies on fluorescence resonance energy transfer (FRET) of hybridization probes. TaqMan probes are oligonucleotides with fluorescent reoprter dye attached to 5′ end and a quenching dye attached to the 3′ end. The probe is designed to hybridize with an internal region of the PCR product. Upon irradiation, the excites reporter dye transfers energy to the quencher dye instead of flourescing. During PCR, when the polymerase enzyme replicates the template that is bound with the probe, the 5′-3′ exonuclease activity of the enzyme cleaves the probe such that the reporter and quencher dyes separate and FRET no longer occurs. This emits fluorescence and PCR products are detected as the probes are cleaved and the templates are amplified. This method is higly effective in detecting PCR products. However, the cost of reagents and equipments is higher than standard PCR experiments.

Applications of PCR:

Amplification and characterization of minute amounts of nucleic acids have revolutionized molecular biology. This method is extremely essential for researchers, medical professionals, and pathologists to identify mutations in human DNA and detect the effects of infectious micro-organisms. Here is a list of major applications of PCR:

• Forensics
• Finger printing and population and analysis
• DNA sequencing
• Molecular cloning
• Archaeology
• Clinical pathology
• Identification and amplification of unknown sequences
• Genetic diagonosis
• Quantitative analysis of DNA or RNA.

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To know in detail about designing primers for PCR reactions, click here.