RESUMO
Fragmentation of synthetic oligonucleotides under the influence of biotin was investigated using 3-hydroxypicolinic acid (3-HPA) as a matrix-assisted laser desorption/ionization (MALDI) matrix. Addition of biotin into the sample enhanced fragmentation of the oligonucleotide between bases. However, when the biotin was tagged to the 5'-terminus of the oligonucleotide, enhancements were observed not only in desorption/ionization efficiency but also in the fragmentation of molecular ions. The protonation/deprotonation process occurs on the tagged biotin is a possible reason for the enhancement in desorption/ionization. Site-specific backbone cleavage fragmentation patterns were observed. The sequences of oligonucleotides can be obtained from their fragment ions. The direct sequencing of a 5'-biotin-tagged 25-mer is demonstrated.
Assuntos
Biotina/química , DNA/química , Análise de Sequência de DNA/métodos , Sequência de Bases , Oligonucleotídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The 5'-3' exonuclease activity of DNA polymerase was utilized in the polymerase chain reaction system to generate a specific signal concomitant with amplification. These signals were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method obviates the need to perform extensive DNA purification of reaction products that is often necessary for detecting larger DNA molecules by mass spectrometry. Oligonucleotides complementary to the internal region of the amplicon are degraded by the 5'-3' exonuclease activity and the degradation products are analyzed by MALDI mass spectrometry. We refer to this assay as the Exo-taq assay or probe degradation assay. This method should be amenable to automation.
Assuntos
DNA/metabolismo , Exonucleases/metabolismo , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Taq Polimerase/metabolismo , DNA/biossíntese , DNA/genética , Humanos , Complexos Multienzimáticos/metabolismoRESUMO
Two new approaches for nucleic acid hybridizations by MALDI-TOF mass spectrometry are described. Hybridization using genomic DNA without polymerase chain reaction was demonstrated. Total genomic DNA of bacteriophages bound to charge-modified nylon membranes was identified by the hybridization of species-specific oligonucleotide probes. lambda-Phage DNA and M13 were used for the test with good success. Since MALDI-TOF mass spectrometry can be used to measure the molecular weights of different probes, mass spectrometry can be used for the detection of hybridizations with multiple probes. We demonstrate that multiple-probe hybridization can be resolved by mass spectrometry. Six probes with different mass tag were used for hybridization on a single spot. MALDI-TOF mass spectrometry was successfully used to measure these probes simultaneously. This provides a simple nonradioactive method for multiplex hybridization analysis. It has the potential to drastically increase the speed for microarray hybridization analysis in the future.
Assuntos
Bacteriófagos/genética , Análise de Sequência de DNA/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Bacteriófago M13/genética , Bacteriófago lambda/genética , DNA Viral/química , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Oligonucleotide ions have been detected using matrix-assisted laser desorption/ionization (MALDI) under nonresonant laser irradiation of the sample. When mass resolution was not limited by adduct attachment to the analyte ions, the nonresonant MALDI spectra demonstrated better resolution than the spectra acquired with resonant ultraviolet irradiation. We found that preparation of thin-film samples on absorbing substrate surfaces was critical for the success of NR-MALDI. The possible acoustic mechanisms of ion formation and desorption are discussed.
Assuntos
Oligonucleotídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria UltravioletaRESUMO
A rapid, simple, and reliable gender determination of human DNA samples was successfully obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Detection sensitivity reached 0.01 ng or less for DNA samples.
Assuntos
DNA/análise , Análise para Determinação do Sexo/métodos , DNA/sangue , DNA/genética , Feminino , Humanos , Masculino , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
In this paper, we report for the first time use of laser desorption mass spectrometry for measurement of chemical cleavage sequencing products of DNA. In this method, the target DNA was labeled with biotin and subjected to chemical modification and cleavage according to the Maxam-Gilbert sequencing protocol. The biotin-containing fragments were captured by streptavidin-coated magnetic beads and separated from the other fragments. The captured fragments were released by hot ammonia treatment, and the released fragments were analyzed by mass spectrometry. Potential applications of this method in resolving sequence ambiguities and sequencing repeat sequences as well as in the analysis of DNA-protein interactions are discussed.
Assuntos
Análise de Sequência de DNA/métodos , Sequência de Aminoácidos , Dados de Sequência Molecular , Oligonucleotídeos/síntese química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Genotyping of the dentatorubral-pallidoluysian atrophy (DRPLA) locus in six patient samples, representing four normal individuals and two DRPLA patients, was successfully obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). DRPLA is a dominantly inherited neurodegenerative disorder associated with the expansion of an unstable trinucleotide (CAG) repeat. The accurate determination of repeat length utilizing MALDI supports the use of this methodology for the analysis of genes containing unstable CAG trinucleotide repeats.
Assuntos
Doenças Neurodegenerativas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Expansão das Repetições de Trinucleotídeos/genética , Alelos , Cromossomos Humanos Par 12 , Genótipo , HumanosRESUMO
Sequencing of DNA fragments of 130 and 200 bp using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for DNA ladder detection was demonstrated. With further improvement in mass resolution and detection sensitivity, mass spectrometry shows great promise for routine DNA sequencing in the future.
Assuntos
Análise de Sequência de DNA/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Bacteriófago lambda/genética , DNA/química , DNA/genética , DNA Viral/química , DNA Viral/genética , Peso Molecular , Sensibilidade e EspecificidadeRESUMO
Matrix-assisted laser desorption/ionization was used for the detection of four base short tandem repeats (STR) for clinical samples using a time-of-flight mass spectrometer. Since STR plays an important role in genetic disease and human identification, this work indicates that laser desorption mass spectrometry has the potential to achieve rapid DNA typing for both forensic applications and genetic disease diagnosis.
Assuntos
DNA/química , Sequências Repetitivas de Ácido Nucleico , DNA/isolamento & purificação , Humanos , Peso Molecular , Mutação , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The DNA sequence of a single-stranded and double-stranded template was determined. The templates were sequenced using the chain termination method and cycle sequencing method and detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The sequencing products were analyzed successfully without the laborious and expensive methods for removal of the template. Direct sequencing of the double-stranded template was achieved with minimal post-reaction purifications, which could be extremely important for mutation analysis and clinical diagnosis. A systematic study of the mechanisms and kinetics of sequencing reactions was also performed. The details of this analysis and directions for future improvements of the quality of sequencing are presented.
Assuntos
DNA de Cadeia Simples/análise , DNA/análise , Análise de Sequência de DNA/métodos , Sequência de Bases , DNA Polimerase Dirigida por DNA , Humanos , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments generated by digestion of a PCR generated target fragment.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Mutação Puntual , Linhagem Celular , Sondas de DNA , Humanos , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric study of DNA detection using 2,3,4-trihydroxyacetophenone, 2,4,6-trihydroxyacetophenone, and their combination has been carried out systematically. The results show that a mixture of 2,4,6-trihydroxyacetophenone, 2,3,4-trihydroxyacetophenone and ammonium citrate with molar ratios of 2:1:1 serves as a good matrix for the detection of DNA, especially for samples containing a small quantity of DNA such as polymerase chain reaction product. The resolution and shot-to-shot reproducibility using this matrix are better than, and the MALDI sensitivity comparable to, that obtained when using 3-hydroxy picotinic acid (3-HPA), PA and ammonium citrate matrix (9:1:1). The mechanism of desorption/ionization using this matrix is discussed.
Assuntos
DNA/análise , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Matrix-assisted laser desorption/ionization (MALDI) was used for several small proteins (such as insulin) and for peptides. It was found that the detection efficiencies of MALDI for the insulin B chain and the insulin A chain are drastically different. Similar phenomena were also observed for various types of peptides. The positive-ion signal of MALDI in detecting proteins or peptides was found to be greatly enhanced by the presence of a basic amino acid in their chains. The experimental results indicate that this enhancement may arise from proton transfer in solution by an acid-base reaction between the protein/peptide and matrix molecule. This pre-protonated mechanism provides a low energy barrier for the ionization of peptides in a MALDI process, and greatly reduces the energy threshold of MALDI. Matrix effects on the ionization mechanism are discussed.
Assuntos
Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Arginina/química , Histidina/química , Insulina/análise , Lisina/química , Dados de Sequência Molecular , Espectrofotometria UltravioletaRESUMO
The most common mutation of the cystic fibrosis gene is characterized by the deletion of three nucleotides that code phenylalanine in the 508 position of the cystic fibrosis transmembrane conductance regulator. We report the first measurements by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry for the delta F508 mutation in cystic fibrosis carriers and patients. Furthermore, in a blind test, results from the normal and delta F508 mutant alleles in 30 clinical samples based on MALDI mass spectrometry and on conventional gel analysis of the DNA were in total agreement. These results demonstrate the utility of MALDI mass spectrometry in the molecular diagnosis of mutant alleles and point to its potential use for ultra-fast detection in large-scale screening of DNA mutations.
Assuntos
Fibrose Cística/genética , DNA/análise , Mutação , Sequência de Bases , Humanos , Lasers , Espectrometria de Massas , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da PolimeraseRESUMO
3-Aminopicolinic acid (3-APA) was tested and found to be a useful matrix for matrix-assisted laser desorption/ionization of DNA and protein. Single-stranded DNA segments of 150-mer and double-stranded DNA of 246 base pairs were successfully detected by using 3-APA as an ultraviolet-absorbing matrix in a linear time-of-flight mass spectrometer. In the case of the double-stranded DNA, only parent ions corresponding to single-stranded DNA were observed. The comparison with 3-hydroxypicolinic acid and picolinic acid matrices is discussed.
Assuntos
DNA/análise , Ácidos Picolínicos/análise , Sequência de Bases , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Oligonucleotídeos/análise , Reação em Cadeia da PolimeraseRESUMO
We found that picolinic acid is a very good matrix for matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry of oligonucleotides, proteins and tRNA. Among the oligonucleotides, picolinic acid was shown to be effective for homo-oligonucleotides, d(G)40 and d(C)60, and for mixed-base oligonucleotides up to 190 bases. In the case of the single-stranded oligonucleotides, and of double-stranded ones as well, only parent ions corresponding to single-stranded DNA were observed. The efficiency for MALDI of oligonucleotides using the picolinic acid matrix was superior to that using 3-hydroxypicolinic acid. MALDI of transfer RNA phenylalanine-specific (tRNA(Phe)), a 76-base ribonucleic acid, was detected with a signal-to-noise ratio of > 10. By comparison with 3-hydroxypicolinic acid the results with picolinic acid are notably superior for all oligonucleotides.
Assuntos
Ácidos Nucleicos/química , Ácidos Picolínicos/química , Proteínas/química , Sequência de Bases , Cristalização , DNA/química , Lasers , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Oligonucleotídeos/química , RNA de Transferência de Fenilalanina/químicaRESUMO
We report the first detection of DNA segments as large as 500 nucleotides by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, using picolinic acid and 3-hydroxypicolinic acid mixtures as desorption matrices. The successful detection of 500-nucleotide DNA indicates that laser mass spectrometry is now emerging as a new biotechnology tool for DNA-related research. It should be possible to utilize fast detection of large DNA segments by laser mass spectrometry for rapid human genome sequencing.
Assuntos
DNA/química , Sequência de Bases , Humanos , Região Variável de Imunoglobulina/genética , Lasers , Espectrometria de Massas , Dados de Sequência Molecular , Ácidos PicolínicosRESUMO
Matrix-assisted laser desorption/ionization, by a time-of-flight mass spectrometer, has been successfully used for detection of restriction enzyme-digested DNA. However, the oligonucleotide segments detected correspond to the molecular weights of single strands.
Assuntos
DNA/análise , Sequência de Bases , Enzimas de Restrição do DNA , DNA de Cadeia Simples/análise , Hidrólise , Lasers , Espectrometria de Massas , Dados de Sequência Molecular , Oligonucleotídeos/análise , Reação em Cadeia da PolimeraseRESUMO
Ferulic acid, 3-hydroxypicolinic acid (3-HPA), and 2,5-dihydroxybenzoic acid (2,5-DHB) were used as matrices in MALDI of various oligonucleotides. It was found that 2,5-DHB is an excellent matrix for polydeoxyribothymidylic acid. A poly-T oligonucleotide with a size of 130 bases was successfully detected. 3-HPA was found to be a good matrix for MALDI of homo-oligomeric deoxynucleotides as well as those containing four different bases. Parent ions of pd(A)60, d(T)100, d(G)40, and d(C)40 were observed, and a mixed-base oligonucleotide of 150-mer was also detected. Polymer ions of poly-A as large as 420-mer were measured; however, the efficiency for detecting poly-T with 3-HPA as a matrix was slightly worse than when 2,5-DHB was used. Comparisons of three matrices for various oligonucleotides is also presented.
Assuntos
Espectrometria de Massas/métodos , Oligonucleotídeos , Sequência de Bases , Dados de Sequência MolecularRESUMO
Various isomer matrices were used for laser-desorption ionization of mixtures of oligomers. It was found that the efficiency of production of oligomer ions changed drastically when different isomers were used as matrices. For selected matrix materials, parent oligomer ions with sizes up to 64 bases were observed.