How it Works: Obtaining Cost-Effective and Precise qPCR Chemistries

Problem: Quantitative PCR (qPCR) assays using the 5’ nuclease process are used for a variety of applications, from basic validation to complex screening studies. The volume of reagents and enzymes used in a given time frame can, therefore, vary extensively depending on the application and research laboratory. Unfortunately, due to restrictions on reaction scales available for purchase, researchers must often pay for larger solution volumes than they will use, especially when analyzing a limited number of samples. This is not cost-effective and is extremely wasteful. Furthermore, the issue of cost may also lead some researchers to choose intercalating fluorescent dye-based methods instead of the 5’ nuclease method which is less specific and may not be able to provide the necessary quality of results. The real-time data obtained may not always be a true representation of the amplification due to the occurrence of primer-dimers and non-specific binding. Researchers can avoid these issues by using the 5’ nuclease-based PrimeTime assays from Integrated DNA Technologies (IDT).

Solution: IDT has recently developed its PrimeTime qPCR assay portfolio. This complete range of assay scales enables more affordable gene studies which better reflect the needs of the customer. By providing quantities that accurately match a broad range of research applications, scientists can work in a cost-effective manner and stay within their allocated budgets. The PrimeTime Mini, consisting of 100 reactions, is ideal for those requiring a limited number of reactions and makes smallerscale qPCR more affordable. The PrimeTime Standard (500 reactions) and PrimeTime XL (2,500 reactions) complete the range by offering larger reaction quantities that reflect the needs of more frequent users who perform qPCR experiments on a regular basis.

In addition to the choices in scale, users also have the ability to set the thermodynamic parameters to match the specific requirements of their assay. The assays are designed to reduce the detection of genomic DNA by spanning exon-exon junctions. Furthermore, the sequence of all primers and probes can be viewed and evaluated prior to any purchase. As a result, all personal and experimental preferences can be met with ease.

To avoid the inaccuracies that come with the use of intercalating fluorescent dyes, all of the Prime- Time qPCR product offerings are 5’ nuclease assays consisting of a forward and a reverse primer along with a dual-labeled probe. This oligonucleotide combination allows for relative or absolute quantification of target sequence within a sample. Unlike intercalating dyes, the probe provides improved specificity by increasing fluorescence only when the target sequence is extended. During the elongation phase of the PCR cycle, the polymerase cleaves the 5’ reporter which allows it to separate from the quencher and emit fluorescence. Therefore, this method avoids misleading results that arise from primer-dimers or non-specific binding and provides increased specificity and precise, repeatable results with every reaction.

For more information, visit www.idtdna.com/ primetime.