The use of fully synthetic oligonucleotides as positive control material has been described recently. However, there were limitations as to the length of the oligonucleotide, which was generally unable to reach more than 130 bases. In many situations, for instance in the case of drug resistance mutations, nucleotide substitutions are often scattered within the target genome and thus require larger fragments as standard templates. Here, we took a novel approach to creating long artificial templates by taking advantage of primer-dimers, which are usually considered a common laboratory problem. The binding of two long artificially synthesized oligos generated a substrate for PCR and was used to create long double-stranded DNA molecules of about 170 bp. In most of the current diagnostic studies (especially based on real time PCR and RCA), 150-240 bp would be sufficient to serve a template controls. However, it is possible to use multiple long oligos to synthesize even longer double-stranded DNA and up to the full length of some important genes, if need be, which is a significant advantage over any similar technologies existing currently.
The most significant advantage of using synthetic oligonucleotides is flexibility. Our approach has proven that the use of laboratory safe artificial templates can be a relatively cost effective, simple and efficient alternative to difficult to acquire material related to an infection or bioterrorism agent. The production of standard templates via the formation of oligo dimers and PCR using synthetic oligos does not require the use of viral or bacterial strains and oligos of any desired sequence can be made commercially. This approach may have the potential to produce a false-positive product due to contamination and therefore caution may be necessary. However, this problem can be solved by the introduction of exclusive restriction endonuclease digestion sites in the artificial template, which would allow for rapid confirmation of a false-positive result. Certain restriction endonuclease (SmaI and BamHI) can be used directly in the PCR buffer (without buffer exchange) and thus the PCR product from the control can be easily and rapidly distinguished from testing samples. In addition, the standard artificial template could be designed to be slightly smaller or larger in size, which would allow for direct visual identification of contamination with the synthetic control.
Overall, this technology may have wide ranging applications and may revolutionize molecular diagnostics and the use of multiplexing assays.
Design and synthesis of long oligos
Long single-stranded oligonucleotides of 89-98 bases were designed, each containing wild-type template or drug resistance mutations. After synthesis, each oligonucleotide was PAGE purified (Sigma-Aldrich, Sydney, Australia). Paired long oligos were designed carrying >20bp complementary sequences at their 3′ ends.