Abstract
Quantitative PCR (qPCR) had broad applications in molecular diagnostics, gene expression studies in medicine and microbiology, and forensic biology. Detection of target amplicons during qPCR is facilitated through the use of a non-specific DNA binding dye (e.g.: SYBR Green) or sequence specific DNA probes (e.g.: TaqManR probes). Regardless of application or detection method, a standard curve must be generated from known quantities of the target sequence. Current methods for creation of a standard template include: genomic DNA, linear PCR amplicons, or cloned target sequences. Each method has inherent advantages and disadvantages. An approach that overcomes the drawbacks of these methods is the construction of a synthetic oligonucleotide of the target sequence. Synthetic oligonucleotides can be purchased in lengths of 125 – 2000 bp. This double-stranded product is affordable and easy to obtain. Using a synthetic oligonucleotide as a DNA standard can be useful for difficult to obtain DNA samples that would be used to create a standard DNA fragment through traditional methods. In this study, we have incorporated a synthetic oligonucleotide as a DNA standard in a qPCR assay, which decreases time, reagents, and cost of creating a standard sequence. Comparisons between the use of a synthetic oligonucleotide and a traditional standard template will be presented. The cost and time savings as well as the reliability with using a synthetic oligonucleotide makes this approach attractive to those developing qPCR assays.