Real-time polymerase chain reaction (rtPCR) or quantitative PCR (qPCR) is used to determine the content of transcripts of the gene of interest in a sample (i.e., the content of messenger RNA). It is one of the approaches to studying the physiological role of enzymes in the effects of environmental factors on the expression level of genes encoding these enzymes. In medicine the quantitative detection of infectious agents’ DNA during treatment allows to get the information on correctness or ineffectiveness of the therapy, and it also helps to predict the exacerbation of the diseases.
The basis of the PCR method is the repeated copying (amplification) in vitro of the pieces of DNA during the process of repeated temperature cycles. Artificially synthesized oligonucleotide primers are used for identification of desired DNA in the sample. Primer design is performed in such a way that they are complementary to terminal regions of the target DNA sequence and thus limit a desired DNA fragment. After each cycle of amplification the newly synthesized DNA strands can serve as templates for DNA polymerase enzyme. This mechanism produces an exponential increase in the number of DNA fragments.
On the first step of qPCR, RNA should be isolated from the object of study. After that RNA is converted into DNA using the reverse transcriptase enzyme (which is the technique called reverse transcription reaction). The obtained cDNA molecules can serve as a target for PCR.
Photobiology school will hold a demonstration (with participation of students) of RNA isolation from Arabidopsis thaliana plants, followed by reverse transcription and qPCR for determination of the expression level of two different genes encoding Carbonic Anhydrases (the enzymes catalyzing the reversible reaction of carbon dioxide hydration) in plant cells.
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