Large-scale determination of SNP allele frequencies in DNA pools using MALDI-TOF mass spectrometry

Werner, Monika; Sych, Michael; Herbon, Nicole; Illig, Thomas; König, Inke R.

doi:10.1002/humu.10094

Cited by 84 publications

(56 citation statements)

References 43 publications

(49 reference statements)

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“…The accuracy and precision of this approach have been studied extensively for estimating allele frequencies in DNA pools by primer extension and MALDI-TOF MS (21)(22)(23)(24)(25). The mass spectrometric analytical challenge is actually less demanding for methylation analysis.…”

Section: Discovery Of Genomic Methylation Sitesmentioning

confidence: 99%

Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

Ehrich

Nelson

Stanssens

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

746

647

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Methylation is one of the major epigenetic processes pivotal to our understanding of carcinogenesis. It is now widely accepted that there is a relationship between DNA methylation, chromatin structure, and human malignancies. DNA methylation is potentially an important clinical marker in cancer molecular diagnostics. Understanding epigenetic modifications in their biological context involves several aspects of DNA methylation analysis. These aspects include the de novo discovery of differentially methylated genes, the analysis of methylation patterns, and the determination of differences in the degree of methylation. Here we present a previously uncharacterized method for high-throughput DNA methylation analysis that utilizes MALDI-TOF mass spectrometry (MS) analysis of base-specifically cleaved amplification products. We use the IGF2͞H19 region to show that a single base-specific cleavage reaction is sufficient to discover methylation sites and to determine methylation ratios within a selected target region. A combination of cleavage reactions enables the complete evaluation of all relevant aspects of DNA methylation, with most CpGs represented in multiple reactions. We successfully applied this technology under high-throughput conditions to quantitatively assess methylation differences between normal and neoplastic lung cancer tissue samples from 48 patients in 47 genes and demonstrate that the quantitative methylation results allow accurate classification of samples according to their histopathology. lung cancer ͉ MALDI-TOF MS ͉ epigenetics

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Section: Discovery Of Genomic Methylation Sitesmentioning

confidence: 99%

Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

Ehrich

Nelson

Stanssens

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

746

647

View full text Add to dashboard Cite

show abstract

“…MassARRAY technology (iPLEXs) constitutes a highly sensitive, high-throughput tool for the genotyping of singlenucleotide polymorphisms [25][26][27][28][29][30] and for mutation screening. [31][32][33] To detect nucleic acid changes, target regions are initially amplified using multiplex PCR and subsequently hybridized to custom-designed primers, then subjected to a single base extension reaction using single mass-modified nucleotides.…”

mentioning

confidence: 99%

High-throughput detection of fusion genes in cancer using the Sequenom MassARRAY platform

Lambros

Wilkerson

Natrajan

et al. 2011

Laboratory Investigation

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Fusion genes have pivotal roles in the development and progression of human cancer and offer potential for rational drug design. Massively parallel sequencing has identified a panoply of in-frame expressed fusion genes, but early reports suggest that the majority of these are present at very low prevalence or are private events. Conventional methods for the identification of recurrent expressed fusion genes in large cohorts of cancers (eg fluorescence in situ hybridization (FISH) and reverse transcriptase PCR (RT-PCR)) are time consuming and prone to artifacts. Here, we describe a novel highthroughput strategy for the detection of recurrent fusion genes in cancer based on the Sequenom MassARRAY platform. Fusion genes were initially identified by massively parallel sequencing of breast cancer cell lines. For each fusion gene, two Sequenom probes were designed. Primary human breast cancers and cancer cell lines were interrogated for 10 fusion genes. Sensitivity, specificity, and predictive values of the MassARRAY method were then determined using FISH and qRT-PCR as the 'gold standard.' By combining two probes per fusion gene, the negative and positive predictive values were 100 and 71.4%, respectively. All fusion genes identified by massively parallel sequencing were accurately detected. No recurrent fusion genes were found. The MassARRAY-based approach described here may, therefore, be employed as a high-throughput screening tool for known fusion genes in human cancer. In keeping with other highly sensitive assays, further refinement of this technique is necessary to reduce the number of false-positive results.

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“…A pooling approach, combined with Sequenom technology to determine allele frequencies in case and control populations, was also shown by Werner et al [48]. The accuracy and reliability of the procedure was determined in pools of eight previously genotyped individuals.…”

Section: Quantificationmentioning

confidence: 97%

Typing of single nucleotide polymorphisms by MALDI mass spectrometry: Principles and diagnostic applications

Sauer

2006

Clinica Chimica Acta

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Background: After the completion of the human genome sequencing project human genetics has now shifted its focus to DNA variation. DNA variation analysis is considered to be a key in partly understanding the mechanisms of complex diseases or varying patient responses in drug treatment. One of the major goals in genetics is finding the DNA variants that can act as diagnostic markers for predisposition to specific diseases. Moreover, in microbiology DNA variation has long been known to help discriminate and identify bacterial strains and viruses. Diagnostics based on DNA or RNA detection might be advantageous as an early-stage indication can be provided. Methods: Many simple and efficient methods for the analysis of nucleic acids are already available. Consequently, the last few years have seen an increased in the use of large-scale analysis of nucleic acids, in basic DNA variation studies along with diagnostics. Mass spectrometry techniques such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) can be of great use for genome variation analysis. In particular high-throughput SNP analysis by MALDI can be performed using fully integrated platforms. Conclusions: Mass spectrometry-based procedures have promise for SNPs analysis especially for clinical diagnostics. D

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Large-scale determination of SNP allele frequencies in DNA pools using MALDI-TOF mass spectrometry

Cited by 84 publications

References 43 publications

Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

High-throughput detection of fusion genes in cancer using the Sequenom MassARRAY platform

Typing of single nucleotide polymorphisms by MALDI mass spectrometry: Principles and diagnostic applications

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