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

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 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. (J Am Soc Mass Spectrom 2002, 13, 232-240)

Analysis of nucleic acids by FTICR MS

“…It has been demonstrated that solution conditions, thermal denaturaturation effects, and the intrinsic hydrophobicity of the individual strands of the PCR product can significantly influence spectral characteristics (Mangrum, Flora, & Muddiman, 2002;Null, Nepomuceno, & Muddiman, 2003). Null and co-workers demonstrated that the ESI-FTICR signal intensity of oligonucleotides containing an alkyl modification on the 5 0 -terminus is enhanced relative to their non-modified counterparts.…”

Leveling response factors in the electrospray ionization process using a heated capillary interface

“…The heated electrospray source also denatured the unmodified SNP 22 and SNP 22 containing 7-deaza guanine (data not shown). The ability of the newly fashioned heated electrospray source to denature amplicons containing 7-deaza analogs is a salient result, especially since the previous heated electrospray source [7] was unable to denature these species (unpublished observations).…”

“…Originally, our laboratory designed a heated electrospray source in an attempt to denature duplex DNA [7]. Herein, we describe a completely redesigned source that is more stable, allowing for broader applications.…”

Dual-micro-ESI source for precise mass determination on a quadrupole time-of-flight mass spectrometer for genomic and proteomic applications

“…Internal calibration for PCR amplicons derived from simple and compound STRs Electrospray solution compositions were adjusted to promote the detection of only single-stranded PCR amplicons for all oligonucleotide spectra [45]. Figure 2 shows ESI-Q-ToF mass spectra obtained for the single-stranded HUMTH01 PCR amplicons generated from a synthetic template ( Fig.…”

“…The resulting average mass error for the 35.6 kDa molecule confirmed that the individual is homozygous for allele 18 rather than allele 18'. It has also been shown that single-stranded PCR amplicons can be generated by increasing the aqueous content of the ESI solution [45].…”