Solution composition and thermal denaturation for the production of single-stranded PCR amplicons: piperidine-induced destabilization of the DNA duplex?
Strategies to produce single-stranded PCR amplicons for detection by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) were investigated using modified electrospray solutions and by thermally denaturing the duplex structures with a resistively heated electrospray ionization source. A synthetic 20-mer oligonucleotide annealed to its complementary strand was used as a model system for initial experiments. Electrospray solutions were altered by varying the relative proportion of aqueous phase in efforts to induce destabilization of the double helix. When the electrospray solution contains a 25% aqueous content, the 20-mer oligonucleotide is detected in its double-stranded form. Increasing the proportion of aqueous phase in the electrospray solution to 60% destabilized the double helix, resulting in the detection of only single-stranded species. This strategy was extended to an 82-bp polymerase chain reaction (PCR) product derived from the human tyrosine hydroxylase gene (HUMTH01). In efforts to destabilize the 82-bp PCR product, electrospray solutions reaching 70% aqueous content were necessary to promote the detection of only single-stranded amplicons. Implementation of the resistively heated transfer line and an electrospray solution in which the oligonucleotide is on the threshold of duplex stability allowed for double-stranded and single-stranded species to be generated from the same ESI solutions at both ambient and elevated transfer line temperatures, respectively, without disruption of the electrospray process. The volatile base piperidine, present at 20 mM concentrations in the electrospray solution, was found to play a critical role in the formation of single-stranded species at the higher aqueous percentages and a duplex destabilization mechanism has been proposed.
Mangrum
John B
JB
Department of Chemistry, Virginia Commonwealth University, Richmond 23284, USA.
Flora
Jason W
JW
Muddiman
David C
DC
D017357:Cyclotrons; D004247:DNA / Q000737:chemistry* / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000737:chemistry; D005583:Fourier Analysis; D006358:Hot Temperature; D006801:Humans; D009691:Nucleic Acid Denaturation / Q000187:drug effects*; D009841:Oligonucleotides / Q000737:chemistry*; D010880:Piperidines / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D010880:Piperidines; D004247:DNA