Quantitative Real-Time Polymerase Chain Reaction

QRT-PCR refers to the method of determining the amount of polymerase chain reaction (PCR) products. Biologists use this method to investigate real-time gene expression, and it is the same as quantitative reverse transcriptase (QRT). Determining genes without quantifying their expression requires QRT-PCR. The information generated in the course of the reaction is used to analyze the quantity of PCR product and to monitor the effects of prime concentration and temperature on the reaction (Dobbelstein, 2011). Reverse transcription is mainly applicable when studying gene expression. The researcher should first extract and convert RNA into cDNA before performing the PCR process.

Normally, determining the ratio of one DNA sample to another follows the procedure A/B, where A refers to the copies of DNA in the first sample, and B is equal to the DNA copies in the second sample. The same samples could be hybridized with other agents to determine specific genes. However, the DNA level could be too low to determine using this procedure. Dobblestein (2011) asserts that PCR amplifies the amount to be detectable. The ratio of DNA in the amplified samples is mounted over a PCR. After the reaction, the PCR products are placed on a gel, which indicates a stronger sample with a darker band and a weaker sample with a lighter band. Amplifying the DNA samples can occur until the PCR product becomes constant. This is the saturation point, where DNA hybridization has reached a climax, blocking primer binding and primer extension.

In case the low amounts of DNA are amplified while the samples with more copies do not reach saturation point, the correct PCR can be determined by distributing its mixes to many tubes and placing them on a conventional PCR machine. After 15 to 30 circles, the samples are mounted on a gel to show the strands in each tube (Dobbelstein, 2011). Real-time monitoring of DNA amplification uses quantitative PCR, which relies on fluorescence. Its main limitation is the amplification of a few samples, which leads to the exclusion of others that might still contain substantial DNA amounts. Moreover, using product ratio to convert amplified DNA is uncertain. The process is prone to inhibitors present in other biological samples.

The main application of qPCR is to quantify nucleic acid molecules in environmental and biological samples. Moreover, QRT-PCR generates quantitative data, with the control point being the corrected ratio of the samples being detected, messenger RNA bands, delta Ct values, and the density of DNA in the experimental sample. In molecular biology research, qPCR is used to amplify specific genetic elements and taxonomic markers obtained from environmental samples. Genetic fragments represent functional units, and amplifying them enables the researcher to determine the amount of that element in the sample before subjecting it to the PCR process (Dobbelstein, 2011). The primary taxonomic markers used to determine the quantity of a sample are ribosomal genes. Detecting genetically modified organisms and phytopathogens, as well as clinical genotyping, exemplify real-life applications of qPCR.

In summary, qPCR is a biological method used to determine and compare low quantities of DNA with a specific sequence. In biological research, the method is instrumental in determining genotypes and detecting phytopathogens and genetically modified organisms. qPCR is accompanied by disparate limitations, including uncertainty following the use of product ratio to quantify amplified DNA. The biological inhibitors present in different DNA samples also limit qPCR.

Proposed question: explain the use of qPCR when cloning expressed genes.

Reference

Dobbelstein, M. (2011). Quantitative real-time PCR (qRT-PCR or qPCR) [Video]. YouTube.