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Biomedical subjects

Dirk van den Boom

Publications and source records attributed to Dirk van den Boom.

At least 19 recordsLinked to original sources
PubMed2006

Cytosine methylation profiles as a molecular marker in non-small cell lung cancer.

Aberrant promoter methylation is frequently observed in different types of lung cancer. Epigenetic modifications are believed to occur before the clinical onset of the disease and hence hold a great promise as early detection markers. Extensive analysis of DNA methylation has been impeded by methods that are either too labor intensive to allow large-scale studies or not sufficiently quantitative to measure subtle changes in the degree of methylation. We used a novel quantitative DNA methylation analysis technology to complete a large-scale cytosine methylation profiling study involving 47 gene promoter regions in 96 lung cancer patients. Each individual contributed a lung cancer specimen and corresponding adjacent normal tissue. The study identified six genes with statistically significant differences in methylation between normal and tumor tissue (P < 10(-6)). We explored the quantitative methylation data using an unsupervised hierarchical clustering algorithm. The data analysis revealed that methylation patterns differentiate normal from tumor tissue. For validation of our approach, we divided the samples to train a classifier and test its performance. We were able to distinguish normal from lung cancer tissue with >95% sensitivity and specificity. These results show that quantitative cytosine methylation profiling can be used to identify molecular classification markers in lung cancer.

Biomarkers, Tumor↗
PubMed2006

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale.

We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.

Adaptation, Physiological↗
PubMed2005

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

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 IGF2/H19 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.

Binding Sites↗
PubMed2005

A single nucleotide polymorphism based approach for the identification and characterization of gene expression modulation using MassARRAY.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. Their abundance and the ease with which they can be assayed have lead to their use in applications beyond simple genotyping. One such application is the quantitative determination of transcript levels associated with distinct alleles or haplotypes found in promoters and coding regions of genes. These changes in expression due to allelic variation are often associated with additional genomic or transcript modifications such as DNA methylation or RNA editing. Here, we describe the use of an integrated genetic analysis platform, based on matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) to first, discover coding SNPs (cSNPs); second, use these cSNPs to identify and analyze allele-specific expression; and third, from this knowledge to further analyze methylation patterns as a putative cause for the allele-specific expression. An established model involving allele-specific expression profiles of the human tumor protein 73 (TP73) gene is presented as an example to outline and validate data obtained from the MassARRAY platform. The availability of a single integrated platform to assay stable and dynamic variation at the genomic and transcript level greatly simplifies complex functional genomic studies.

5' Untranslated Regions↗
PubMed2005

Multiplexed discovery of sequence polymorphisms using base-specific cleavage and MALDI-TOF MS.

The completion of the Human Genome Project provides researchers with a reference sequence that covers about 99% of the gene-containing regions and is more than 99.9% accurate. Sequence drafts and completed sequences for several other species are also available to researchers worldwide. The ongoing effort to provide more and more genomic reference information now enables the detection of deviations from this 'genetic blueprint'. Comparative sequencing projects will play a major role in elucidating the meaning of the genetic code and in establishing a correlation between genotype and phenotype. As part of this effort, a number of projects will focus on distinct functional aspects, like resequencing of exons or HLA determining regions. Typically these target regions are short in length and their analysis does not require long read length. To find an efficient solution for these applications, we developed a novel method that allows simultaneous analysis of multiple independent target regions (Multiplexed Comparative Sequence Analysis) by employing base-specific cleavage biochemistry and MALDI TOF-MS analysis.

Humans↗
PubMed2005

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Major strengths of mass spectrometry analysis include the accuracy of the detection principle and the automatic data storage, making it a premier choice for high-throughput single-nucleotide polymorphism genotyping. We explain the assay principle in detail and give step-by-step laboratory instructions. Finally, references point toward further use of mass spectrometry analysis for molecular haplotyping, pooled DNA analysis, re-sequencing, and gene expression studies.

Automation↗
PubMed2004

High-throughput MALDI-TOF discovery of genomic sequence polymorphisms.

We describe a comparative sequencing strategy that is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of complete base-specific cleavage reactions of a target sequence. The target is converted to a DNA/RNA mosaic structure after PCR amplification using in vitro transcription. Cleavage with defined specificity is achieved by ribonucleases. The set of cleavage products is subjected to mass spectrometry without prior fractionation. The presented resequencing assay is particularly useful for single-nucleotide polymorphism (SNP) discovery. The combination of mass spectra from four complementary cleavage reactions detects approximately 98% of all possible homozygous and heterozygous SNPs in target sequences with a length of up to 500 bases. In general, both the identity and location of the sequence variation are determined. This was exemplified by the discovery of SNPs in the human gene coding for the cholesteryl ester transfer protein using a panel of 96 genomic DNAs.

Carrier Proteins↗
PubMed2004

High-throughput mutation detection underlying adaptive evolution of Escherichia coli-K12.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of base-specific cleavage products is an efficient, highly accurate tool for the detection of single base sequence variations. We describe the first application of this comparative sequencing strategy for automated high-throughput mutation detection in microbial genomes. The method was applied to identify DNA sequence changes that occurred in Escherichia coli K-12 MG1655 during laboratory adaptive evolution to new optimal growth phenotypes. Experiments were based on a genome-scale in silico model of E. coli metabolism and growth. This model computes several phenotypic functions and predicts optimal growth rates. To identify mutations underlying a 40-d adaptive laboratory evolution on glycerol, we resequenced 4.4% of the E. coli-K12 MG1655 genome in several clones picked at the end of the evolutionary process. The 1.54-Mb screen was completed in 13.5 h. This resequencing study is the largest reported by MALDI-TOF mass spectrometry to date. Ten mutations in 40 clones and three deviations from the reference sequence were detected. Mutations were predominantly found within the glycerol kinase gene. Functional characterization of the most prominent mutation shows its metabolic impact on the process of adaptive evolution. All sequence changes were independently confirmed by genotyping and Sanger-sequencing. We demonstrate that comparative sequencing by base-specific cleavage and MALDI-TOF mass spectrometry is an automated, fast, and highly accurate alternative to capillary sequencing.

Adaptation, Biological↗
PubMed2004

Novel mass spectrometry-based tool for genotypic identification of mycobacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR amplified and in vitro-transcribed 16S rRNA gene (rDNA) was used for the identification of mycobacteria. Full-length 16S rDNA reference sequences of 12 type strains of Mycobacterium spp. frequently isolated from clinical specimens were determined by PCR, cloning, and sequencing. For MALDI-TOF MS-based comparative sequence analysis, mycobacterial 16S rDNA signature sequences ( approximately 500 bp) of the 12 type strains and 24 clinical isolates were PCR amplified using RNA promoter-tagged forward primers. T7 RNA polymerase-mediated transcription of forward strands in the presence of 5-methyl ribo-CTP maximized mass differences of fragments generated by base-specific cleavage. In vitro transcripts were subsequently treated with RNase T1, resulting in G-specific cleavage. Sample analysis by MALDI-TOF MS showed a specific mass signal pattern for each of the 12 type strains, allowing unambiguous identification. All 24 clinical isolates were identified unequivocally by comparing their detected mass signal pattern to the reference sequence-derived in silico pattern of the type strains and to the in silico mass patterns of published 16S rDNA sequences. A 16S rDNA microheterogeneity of the Mycobacterium xenopi type strain (DSM 43995) was detected by MALDI-TOF MS and later confirmed by Sanger dideoxy sequencing. In conclusion, analysis of 16S rDNA amplicons by MS after base-specific cleavage of RNA transcripts allowed fast and reliable identification of the Mycobacterium tuberculosis complex and ubiquitous mycobacteria (mycobacteria other than tuberculosis). The technology delivers an open platform for high-throughput microbial identification on the basis of any specific genotypic marker region.

Genotype↗
PubMed2004

MALDI-TOF mass spectrometry: a versatile tool for high-performance DNA analysis.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has developed during the past decade into a versatile tool for biopolymer analysis. The aim of this review is to summarize this development and outline the applications, which have been enabled for routine use in the field of nucleic acid analysis. These include the analysis of mutations, the resequencing of amplicons with a known reference sequence, and the quantitative analysis of gene expression and allelic frequencies in complex DNA mixtures.

Animals↗
PubMed2003

RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis.

Here we devise a new method for high-throughput comparative sequence analysis. The developed protocol comprises a homogeneous in vitro transcription/RNase cleavage system with the accuracy and data acquisition speed of matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS). In summary, the target region is PCR amplified using primers tagged with promoter sequences of T7 or SP6 RNA polymerase. Using RNase T1, the in vitro transcripts are base-specifically cleaved at every G-position. This reaction results in a characteristic pattern of fragment masses that is indicative of the original target sequence. To enable high-throughput analysis, samples are processed with automated liquid handling devices and nanoliter amounts are dispensed onto SpectroCHIP arrays for reliable and homogeneous MALDI preparation. This system enables rapid automated comparative sequence analysis for PCR products up to 1 kb in length. We demonstrate the feasibility of the devised method for analysis of single nucleotide polymorphisms (SNPs) and pathogen identification.

Apolipoproteins B↗
PubMed2003

High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases.

We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.

Asthma↗
PubMed2002

Association testing by DNA pooling: an effective initial screen.

With an ever-increasing resource of validated single-nucleotide polymorphisms (SNPs), the limiting factors in genome-wide association analysis have become genotyping capacity and the availability of DNA. We provide a proof of concept of the use of pooled DNA as a means of efficiently screening SNPs and prioritizing them for further study. This approach reduces the final number of SNPs that undergo full, sample-by-sample genotyping as well as the quantity of DNA used overall. We have examined 15 SNPs in the cholesteryl ester transfer protein (CETP) gene, a gene previously demonstrated to be associated with serum high-density lipoprotein cholesterol levels. The SNPs were amplified in two pools of DNA derived from groups of individuals with extremely high and extremely low serum high-density lipoprotein cholesterol levels, respectively. P values <0.05 were obtained for 14 SNPs, supporting the described association. Genotyping of the individual samples showed that the average margin of error in frequency estimate was approximately 4% when pools were used. These findings clearly demonstrate the potential of pooling techniques and their associated technologies as an initial screen in the search for genetic associations.

Adolescent↗
PubMed2002

Base-specific fragmentation of amplified 16S rRNA genes analyzed by mass spectrometry: a tool for rapid bacterial identification.

A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNA-glycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yet-uncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria.

Bacterial Typing Techniques↗
PubMed2002

The use of MassARRAY technology for high throughput genotyping.

This chapter will explore the role of mass spectrometry (MS) as a detection method for genotyping applications and will illustrate how MS evolved from an expert-user-technology to a routine laboratory method in biological sciences. The main focus will be time-of-flight (TOF) based devices and their use for analyzing single-nucleotide-polymorphisms (SNPs, pronounced snips). The first section will describe the evolution of the use of MS in the field of bioanalytical sciences and the protocols used during the early days of bioanalytical MALDI TOF mass spectrometry. The second section will provide an overview on intraspecies sequence diversity and the nature and importance of SNPs for the genomic sciences. This is followed by an exploration of the special and advantageous features of mass spectrometry as the key technology in modern bioanalytical sciences in the third chapter. Finally, the fourth section will describe the MassARRAY technology as an advanced system for automated high-throughput analysis of SNPs.

Databases, Nucleic Acid↗