Analysis of short tandem repeat polymorphisms by electrospray ion trap mass spectrometry

Abstract

The application of electrospray ionization (ESI) ion trap mass spectrometry (MS) to the analysis of short tandem repeats (STRs or microsatellites) is described. Several equine dinucleotide STR loci were chosen as a model system to evaluate ESI ion trap as a routine instrument for rapid and reliable genoytping. With the use of specific primers STR loci were amplified from different blood samples having allele sizes between 60 and 100 bp. A new purification method based on reversible binding of PCR products to magnetic particles has proven to be directly compatible with ESI ion trap MS analysis. The sense and antisense strands of the PCR products with concentrations of ∼100 fmol/µl were measured with a mass accuracy of 0.01%. The simplicity of the purification method and the capability for automated handling together with the precise sizing of PCR products by ESI ion trap MS facilitate the large scale analysis of polymorphic STRs. Moreover, mixtures of different allele length as obtained for heterozygous samples could accurately be assigned as well as a C→G switch between the two strands of a PCR product.

Evaluation of sample preparation techniques for mass measurements of PCR products using ESI-FT-ICR mass spectrometry

“…This result indicates that the PCR product is not being lost during cleanup, but that the magnetic beads are not efficient enough to observe a PCR product in the mass spectrum. This result is inconsistent with a previous report using Genopure ds [14]. Figure 1b shows a single-acquisition ESI-FT-ICR mass spectrum of the single-reaction, double-stranded PCR product derived from the HUMTH01 locus using GENECLEAN followed by an ethanol precipitation.…”

“…The PCR product was eluted in 10 L of water. This approach has recently been shown for PCR products using ESI ion trap mass spectrometry [14].…”

“…Numerous techniques have been utilized for the preparation of oligonucleotides and PCR products for mass spectrometry including immobilization by biotin/ streptavidin chemistry [8 -10], phenol/chloroform extractions [11,12], size-exclusion filters [8,[12][13][14], silica resin [10], cation-exchange resin beads [15], anionexchange HPLC [15], C 18 purification pipet tips [15], desalting columns [15], minidialysis [15], gel filtration [16], magnetic particles [14], ethanol precipitation [10, 11, 14, 16 -18], and microdialysis [19 -21]. The coupling of an ethanol precipitation and microdialysis, first demonstrated by our group [22], has been shown to efficiently desalt PCR products for analysis by mass spectrometry [4,6,[22][23][24][25][26][27][28].…”

Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry

“…Commonly applied off-line and on-line sample purification protocols for nucleic acids prior to mass spectrometric analysis include multiple ethanol precipitation [19], membrane filtration [20], solid-phase extraction [21], microdialysis [14], affinity purification [22,23], cation-exchange [24], ligand-exchange [13], magnetic particles [25], size exclusion chromatography [26], and high-performance liquid chromatography (HPLC) [27,28]. Ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization mass spectrometry (ICEMS) has been demonstrated to be a rapid and highly effective analytical tool for the characterization of PCR amplified sequences [15, 29 -31].…”

Allelic loss analysis by denaturing high-performance liquid chromatography and electrospray ionization mass spectrometry

“…This continues to spur the development of new algorithms to reduce errors and human intervention in electrophoretic genotyping [Johansson et al, 2003] and of novel methodology offering higher precision. Foremost among alternative approaches to STR genotyping have been various mass spectrometric methods, including EI Fourier transform ion cyclotron resonance MS [Null et al, 2001], ESI-IT-MS [Hahner et al, 2000;Oberacher et al, 2001b], and matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) MS [Taranenko et al, 1999;Wada et al, 1999]. However, to date these techniques have been only applicable to relatively short DNA fragments of r120 bp [Butler and Becker, 2001], severely limiting their utility and requiring the redesign of amplification primers, which is not always feasible due to the presence of paralogs in the genome.…”