RESUMEN
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.
Asunto(s)
Genotipo , Polimorfismo de Nucleótido Simple , Regiones no Traducidas 5' , Alelos , Metilación de ADN , Proteínas de Unión al ADN/genética , Expresión Génica , Genes Supresores de Tumor , Humanos , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Proteína Tumoral p73 , Proteínas Supresoras de TumorRESUMEN
To find genes that underlie disease susceptibilities, genome-wide single nucleotide polymorphisms (SNPs) have been analyzed using high-throughput matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). As a proof-of-concept for this approach, gene regions have been identified that were previously associated by others with certain diseases or traits. On the same technology platform, accurate and absolute transcriptional profiling can be performed and applied to allele expression analysis. Here, we provide a brief review of the technology and its applications to disease gene discovery.
Asunto(s)
Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Proteoma , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Alelos , Quimiocinas/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Genoma , Humanos , Farmacogenética , Reacción en Cadena de la Polimerasa , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Análisis Mutacional de ADN/métodos , ADN/análisis , ADN/química , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ADN/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Secuencia de Bases , ADN/genética , Humanos , Repeticiones de Microsatélite/genética , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
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.
Asunto(s)
Guanina/metabolismo , Ribonucleasa T1/metabolismo , Análisis de Secuencia de ADN/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Apolipoproteínas B/genética , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/instrumentación , Análisis de Secuencia de ADN/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Bases de Datos de Ácidos Nucleicos , Diseño de Equipo , Proyecto Genoma Humano , Humanos , Polimorfismo de Longitud del Fragmento de Restricción , Especificidad de la Especie , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/tendencias , Secuencias Repetidas en Tándem/genéticaRESUMEN
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.