Working in real time: A focus on qPCR

/Working in real time: A focus on qPCR

Working in real time: A focus on qPCR

There are lots of different types of PCR (polymerase chain reaction), with many different applications, so what’s so special about quantitative PCR (qPCR), also known as real time PCR, RT PCR or qRT-PCR?

Measuring in real time

Whereas conventional PCR focuses on amplifying DNA, and the results are not seen until the end of the process, qPCR is able to quantify the levels of amplification of the target DNA strands (amplicons) precisely, and follow this amplification in ‘real time’.

The qPCR process uses fluorescent reporter molecules or probes that bind specifically or non-specifically to the amplicons. The rising level of florescence therefore correlates directly with the increasing numbers of amplicons in each PCR cycle, and this can be used to report the precise concentration of amplicons. The rate of increase in fluorescence will depend on how much template DNA was included at the start of the process.

There are a number of different categories of probes – exonuclease probes, hybridisation probes, stem-loop probes and intercalating dyes:

  • Exonuclease probes combine a quencher molecule and a reporter molecule (fluorophore). The probes bind specifically to a target sequence on the amplified DNA and then are cleaved by the DNA polymerase, which moves the dye and quencher apart and allows the dye to fluoresce.
  • Hybridisation probes bind specifically and produce different levels of fluorescence according to the levels of mismatches between the probe and the target DNA sequence, and can therefore be used to differentiate between different alleles (different forms of the same gene)
  • Stem-loop or hairpin probes have a fluorescent dye at one end and a quencher at the other. They fold back on themselves, forming a loop that can bind specifically to a target, with the dye and quencher next to each other at the end of the ‘stem’. On hybridisation, the dye and quencher move apart and the dye fluoresces.
  • Intercalating dyes do not show much fluorescence in solution, but fluoresce when they bind to the grooves in double-stranded DNA. These are non-specific, and will bind to any double-stranded DNA, including non-specific PCR products.

Using different fluorophores for different genetic sequences allows the user to quantify a number of genes in a single reaction.

The advantages of qPCR

The key advantage of qPCR is in its name – that it is quantitative, and this quantification can be followed in real time in each cycle, during the early and exponential phases of the process, and the levels of the DNA or RNA can be detected in each cycle, relative to the amounts of DNA or RNA template used at the beginning of the reaction. As qPCR combines amplification and detection in the same process, this reduces the risk of contamination or experimental error.

Uses for qPCR

The qPCR process has a range of applications, including:

  • Gene quantification, including gene deletion and gene duplication
  • Gene and miRNA expression profiling analysis
  • Genotyping and allelic discrimination
  • Somatic mutation analysis
  • Copy number detection and variation analysis
  • Pathogen detection and quantification
  • Discovering biomarkers
  • Validation of microarrays
  • Validation of drug targets
  • Optimisation of DNA- and protein-based biopharmaceuticals
  • Testing of cell lines and cell banks
  • Potency assays of DNA vaccines

Suzanne Elvidge is a freelance science, biopharma, business and health writer with more than 20 years of experience. She is editor of Genome Engineering, a blog that monitors the latest developments in genome engineering and that aims to educate (and sometimes to entertain!) and has written for a range of online and print publications including FierceBiomarkers, FierceDrugDelivery, European Life Science, the Journal of Life Sciences (now the Burrill Report), In Vivo, Life Science Leader, Nature Biotechnology, PR Week and Start-Up. She specialises in writing on pharmaceuticals, biotechnology, healthcare, science, lifestyle and green living, but can write on any topic given enough tea and chocolate biscuits. She lives just beyond the neck end of nowhere in the Peak District with her second-hand bookseller husband and two second-hand cats.

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