You’re conducting RT-qPCR in order to examine levels of gene expression of PAL5
ID: 97105 • Letter: Y
Question
You’re conducting RT-qPCR in order to examine levels of gene expression of PAL5 in your tomato plant. The “q” is for quantitative and the “RT” is for reverse transcriptase, and “PCR” is polymerase chain reaction, which amplified DNA or cDNA.
Why is the enzyme, reverse transcriptase being used in this procedure? What will it do?
What is the original molecule we’re trying quantifying by using RT-qPCR?
What does the enzyme, DNA polymerase do in this reaction?
Primers were designed to amplify a small section PAL5 gene (the gene we’re trying to quantify) as well a probe (24 nucleotides). Primers were also designed to amplify a small section of the a-tubulin gene (which is our housekeeping gene, or constitutive gene) as well a probe (25 nucleotides). In order for RT-qPCR to work, both the primers and the probes will anneal to the cDNA with complementarity.
How are these primers and probes going to anneal, meaning what kind of bonds are being made for this binding to occur?
Explanation / Answer
- In the process of RT-qPCR the sample is RNA which is first converted into DNA by using reverse transciptase and then DNA is amplified by traditional PCR technique. Here we are examing levels of gene expression of PAL5 gene. For this we will have to collect the transcribed mRNA from PAL5 gene. (Since gene express itself via the formation of mRNA). Now our sample is mRNA which is required to convert into their complimentry DNA for further cloning through traditional PCR. Hence reverse transciptase is required.
- The original molecule (sample) for RT-qPCR is mRNA trranscribed from the gene PAL5.
- DNA polymerase is the essential component of PCR technique. DNA polymerase synthesizes the new copy of DNA complimentry to the target DNA. The polymerase begin synthesizing new DNA from the end of the primer (short pieces of single stranded DNA which is complimentry to the target DNA sequence). Hence DNA polymerase synthesizes new copy of target DNA molecule by using RNA primer, nucleotides (A, T, C, G) and DNA template.
- PCR technique involves three steps: Denaturation, Annealing and extension. In the first step the temperature is raised to approximately 94 degrre F. which causes breaking of hydrogen bonds between two strands of DNA due to which unwinding and separation of strands occurs. Next step is annealing in which temperature is lowered which allow the attachment of RNA primers to singular strand by reforming gydrogen bonds. Hence Hydrogen bonds are involved in this binding process.
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.