PCR cycle consists of three elements: denaturation, annealing, and extension.
At the beginning of each cycle, the two complementary DNA template strands are separated at high temperatures (94°C–95°C) in a process called denaturation.
The temperature is then decreased to allow annealing between the oligonucleotide primers and the template. The temperature for annealing is usually 3°C–5°C.
Optimal temperature for DNA polymerase is reached, thus allowing for DNA replication (extension).
Thermal Cycler: Instrument that carries out PCR amplification.
Central dogma: The pathway for the flow of genetic information.
With RNA synthesis or transcription, the process is carried out using the DNA as a template.
RNA chains can be used as templates for the synthesis of a DNA strand of complementary sequence, in which the end product is referred to as complementary DNA (cDNA).
Protein synthesis, also known as translation, is directed by an RNA template.
Reverse Transcription: The flow of genetic information from RNA to DNA.
Reverse transcription is carried out by a reverse transcriptase that forms the basis of reverse transcriptase PCR.
It is highly sensitive and can be used to detect very small quantities of mRNA. It can be utilized to measure levels of gene expression even when the RNA of interest is expressed at very low levels.
During an RT-PCR process, a single-stranded cDNA is synthesized from a template mRNA using reverse transcription. The cDNA is then amplified by PCR with a pair of oligonucleotide primers corresponding to a specific sequence in the cDNA,
Two strategies of RT-PCR exist: a one-step and a two-step RT-PCR.
Two types of PCR methods can be utilized for analyzing amplified products.
The hot-start PCR strategy is typically used to increase sensitivity, specificity, and yield.
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