Single-nucleotide polymorphisms: analysis by mass spectrometry

Abstract

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry has evolved as a powerful method for analyzing nucleic acids. Here we provide protocols for genotyping single-nucleotide polymorphisms (SNPs) by MALDI based on PCR and primer extension to generate allele-specific products. Furthermore, we present three different approaches for sample preparation of primer-extension products before MALDI analysis and discuss their potential areas of application. The first approach, the 'GOOD' assay, is a purificationfree procedure that uses DNA-modification chemistry, including alkylation of phosphorothioate linkages in the extension primers. The other two approaches use either solid-phase extraction or microarray purification for the purification of primer-extension products. Depending on the reaction steps of the various approaches, the protocols take about 6-8 hours. INTRODUCTIONMatrix-assisted laser desorption-ionization (MALDI) mass spectrometry has become an important, versatile tool in life science 1,2 ( Fig. 1). Mass spectrometry detects the mass/charge ratio (m z -1 ) of analyte ions in positive-ion mode or in negative-ion mode and produces highly accurate data 1,3 . MALDI mass spectrometry is efficiently used for quality control of oligonucleotides and for the analysis of genetic markers such as single-nucleotide polymorphisms (SNPs) 4 . MALDI-based SNP-genotyping procedures can be easily extended to the detection of cytosine methylation in genomic DNA as well as to the analysis of allele-specific expression and detection of alternative splicing 3 . In genotyping, mass spectrometry offers a distinct advantage over fluorescence-based methods in that the unambiguous determination of marker alleles relies on the direct measurement of the molecular weight of allele-specific products. Before MALDI, the products of SNPs are generated by a molecular biology procedure consisting of PCR amplification of the SNP positions of interest, digestion of residual dNTPs by phosphatase treatment, and an allele-specific primer-extension reaction 5,6 .Because experimental molecular biology reactions are done in buffered solutions that contain large amounts of salts, it is necessary to include a step to eliminate those from the sample preparation. Phosphate residues of nucleic acids are a problem for MALDI detection because they provide a site of negative charge in solution. In particular, alkali metal ions such as sodium and potassium interfere with the ionization process, which weakens the analysis. Moreover, chemicals such as urea or guanidine-HCl and detergents perturb matrix crystallization and decrease signals in MALDI. Suitable preparation procedure steps must be taken before MALDI to circumvent those problems and to obtain high-quality mass spectra.Many procedures for the detection of SNPs have been developed. The mass spectrometry companies Sequenom and Bruker Daltonics offer products tailored to SNP 'typing' applications. More information about their respective protocols is available on the Sequenom and Bruker Da...

Pterostilbene 4′‐β‐Glucoside Protects against DSS‐Induced Colitis via Induction of Tristetraprolin

“…The upregulation of TTP mRNA can be induced by arthritis, autoimmune dysfunction, and myeloid hyperplasia, demonstrating the importance of TTP in limiting the inflammatory response [27]. During inflammation, TTP causes mRNA decay of several proinflammatory genes, such as TNF- α , IL-6, CCL2, and CCL3 [28]. Also, TTP has a critical effect on the anti-inflammatory response in DSS-induced colitis [29].…”

The essence on mass spectrometry based microbial diagnostics

“…For example, bacteria can be identified to the species level by protein mass pattern detection, while using the same instrument more in-depth information, if necessary, can be gained by additional mass analysis of their nucleic acids. PCR amplification and MALDI-detection based SNP-genotyping [38][39][40], MALDI-based re-sequencing [41 ,42] or electrospray ionisation mass spectrometry detection of PCR products [43,44] are new alternatives for the analysis of nucleic-acids based microbial markers, which may allow efficient monitoring of microbial epidemics [45]. In contrast to protein profiles, mass spectrometry based sequence analysis of microbial genomes can in principle rapidly and accurately reveal known or emerging antibiotic resistance mediated by gene mutations, mobile genetic elements or virulence factors, and detect viruses.…”

MALDI-TOF MS: Its Application in the Clinical Laboratory and a Paradigm Shift in Clinical Microbiology

“…The advantages in terms of the speed and cost of analyses provide a strong incentive for further developments and improvements for extending the range of MALDI-TOF MS applications. [45]; however, these fail to employ one of the key strengths of MS, the direct mass analysis of PCR products.…”