Solution composition and thermal denaturation for the production of single-stranded PCR amplicons: piperidine-induced destabilization of the DNA duplex?

Journal of the American Society for Mass Spectrometry
John B MangrumDavid C Muddiman

Abstract

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 resisti...Continue Reading

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Citations

May 3, 2005·Journal of the American Society for Mass Spectrometry·Jennifer L FrahmMichael J Burke
Mar 11, 2011·Bioanalysis·William D van Dongen, Wilfried M A Niessen
Dec 16, 2003·Rapid Communications in Mass Spectrometry : RCM·Allison P NullDavid C Muddiman
Sep 25, 2004·Mass Spectrometry Reviews·Steven A HofstadlerJames C Hannis
Jul 19, 2002·Journal of Mass Spectrometry : JMS
May 25, 2005·Rapid Communications in Mass Spectrometry : RCM·Michael BeverlyLynn Machemer
Sep 3, 2008·Rapid Communications in Mass Spectrometry : RCM·Liang ZhuRenato Zenobi
Aug 16, 2003·Analytical Chemistry·Justin L P BeneschCarol V Robinson
Apr 15, 2004·Journal of the American Chemical Society·Rambod DaneshfarJohn S Klassen

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