RESUMEN
OBJECTIVE: The objective of this study was to identify differentially expressed salivary proteins in bisphosphonate-related osteonecrosis of the jaw (BRONJ) patients that could serve as biomarkers for BRONJ diagnosis. SUBJECTS AND METHODS: Whole saliva obtained from 20 BRONJ patients and 20 controls were pooled within groups. The samples were analyzed using iTRAQ-labeled two-dimensional liquid chromatography-tandem mass spectrometry. RESULTS: Overall, 1340 proteins were identified. Of these, biomarker candidates were selected based on P-value (<0.001), changes in protein expression (≥1.5-fold increase or decrease), and unique peptides identified (≥2). Three comparisons made between BRONJ and control patients identified 200 proteins to be differentially expressed in BRONJ patients. A majority of these proteins were predicted to have a role in drug metabolism and immunological and dermatological diseases. Of all the differentially expressed proteins, we selected metalloproteinase-9 and desmoplakin for further validation. Immunoassays confirmed increased expression of metalloproteinase-9 in individual saliva (P = 0.048) and serum samples (P = 0.05) of BRONJ patients. Desmoplakin was undetectable in saliva. However, desmoplakin levels tended to be lower in BRONJ serum than controls (P = 0.157). CONCLUSIONS: Multiple pathological reactions are involved in BRONJ development. One or more proteins identified by this study may prove to be useful biomarkers for BRONJ diagnosis. The role of metalloproteinase-9 and desmoplakin in BRONJ requires further investigation.
Asunto(s)
Osteonecrosis de los Maxilares Asociada a Difosfonatos/diagnóstico , Proteínas/análisis , Saliva/química , Biomarcadores/análisis , Osteonecrosis de los Maxilares Asociada a Difosfonatos/metabolismo , Estudios de Casos y Controles , Cromatografía Liquida , Desmoplaquinas/análisis , Femenino , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/análisis , Persona de Mediana Edad , Proteómica , Espectrometría de Masas en TándemRESUMEN
Pulmonary involvement occurs in up to 95% of sarcoidosis cases. In this pilot study, we examine lung compartment-specific protein expression to identify pathways linked to development and progression of pulmonary sarcoidosis. We characterized bronchoalveolar lavage (BAL) cells and fluid (BALF) proteins in recently diagnosed sarcoidosis cases. We identified 4,306 proteins in BAL cells, of which 272 proteins were differentially expressed in sarcoidosis compared to controls. These proteins map to novel pathways such as integrin-linked kinase and IL-8 signaling and previously implicated pathways in sarcoidosis, including phagosome maturation, clathrin-mediated endocytic signaling and redox balance. In the BALF, the differentially expressed proteins map to several pathways identified in the BAL cells. The differentially expressed BALF proteins also map to aryl hydrocarbon signaling, communication between innate and adaptive immune response, integrin, PTEN and phospholipase C signaling, serotonin and tryptophan metabolism, autophagy, and B cell receptor signaling. Additional pathways that were different between progressive and non-progressive sarcoidosis in the BALF included CD28 signaling and PFKFB4 signaling. Our studies demonstrate the power of contemporary proteomics to reveal novel mechanisms operational in sarcoidosis. Application of our workflows in well-phenotyped large cohorts maybe beneficial to identify biomarkers for diagnosis and prognosis and therapeutically tenable molecular mechanisms.
Asunto(s)
Progresión de la Enfermedad , Proteínas/metabolismo , Sarcoidosis Pulmonar/metabolismo , Adulto , Líquido del Lavado Bronquioalveolar/química , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Proyectos Piloto , Sarcoidosis Pulmonar/patologíaRESUMEN
The ability to analyze multiple polymorphic sites rapidly and accurately is crucial in all areas of genetic analysis. We have developed an approach for the detection of multiple point mutations, using allele-specific, mass-labeled, peptide nucleic acid (PNA) hybridization probes, and direct analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The composite mass spectra produced contain peaks of distinct masses corresponding to each allele present, resulting in a mass spectral "fingerprint" for each DNA sample. The hybridization characteristics of PNA:DNA duplexes were found to be highly dependent on both base content and sequence. Results from the analysis of four polymorphic sites contained in exon 4 of the human tyrosinase gene show that this approach is simple, rapid, and accurate with potential applications in many areas of genetic analysis.
Asunto(s)
ADN/análisis , Monofenol Monooxigenasa/genética , Sondas de Ácido Nucleico/análisis , Ácidos Nucleicos/análisis , Fragmentos de Péptidos/análisis , Mutación Puntual/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , ADN/metabolismo , Exones , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Ácidos Nucleicos/genética , Fragmentos de Péptidos/genética , Reacción en Cadena de la Polimerasa , Polimorfismo GenéticoRESUMEN
We have explored the potential of the Tn 552 in vitro transposition reaction as a genetic tool. The reaction is simple (requiring a single protein component), robust and efficient, readily producing insertions into several percent of target DNA. Most importantly, Tn 552 insertions in vitro appear to be essentially random. Extensive analyses indicate that the transposon exhibits no significant regional or sequence specificity for target DNA and leaves no discernible 'cold' spots devoid of insertions. The utility of the in vitro reaction for DNA sequencing was demonstrated with a cosmid containing the Mycobacterium smegmatis recBCD gene cluster. The nucleotide sequence of the entire operon was determined using 71 independent Tn 552 insertions, which generated over 13.5 kb of unique sequence and simultaneously provided a comprehensive collection of insertion mutants. The relatively short ends of Tn 552 make construction of novel transposons a simple process and we describe several useful derivatives. The data presented suggest that Tn 552 transposition is a valuable addition to the arsenal of tools available for molecular biology and genomics.
Asunto(s)
Elementos Transponibles de ADN , Mutagénesis Insercional , Análisis de Secuencia de ADN/métodos , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/genética , Datos de Secuencia Molecular , Mycobacterium smegmatis/genética , TransposasasRESUMEN
Proteome characterization using mass spectrometry is essential for the systematic investigation of biological systems and for the study of gene function. Recent advances in this multifaceted field have occurred in four general areas: protein and peptide separation methodologies; selective labeling chemistries for quantitative measurement of peptide and protein abundances; characterization of post-translational protein modifications; and instrumentation.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas/análisis , Proteínas/química , Cromatografía Líquida de Alta Presión , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Humanos , Espectrometría de Masas/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos/químicaRESUMEN
Single-nucleotide polymorphisms (SNPs) have great potential for use in genetic-mapping studies, which locate and characterize genes that are important in human disease and biological function. For SNPs to realize their full potential in genetic analysis, thousands of different SNP loci must be screened in a rapid, accurate and cost-effective manner. Matrix-assisted laser desorption-ionization-time-of-flight (MALDI-TOF) mass spectrometry is a promising tool for the high-throughput screening of SNPs, with future prospects for use in genetic analysis.
Asunto(s)
Polimorfismo de Nucleótido Simple , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Genotipo , HumanosRESUMEN
The isotope-coded affinity tag (ICAT) technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.
Asunto(s)
Proteínas/química , Proteoma/química , Autoanálisis , Línea Celular , Células Epiteliales/química , Humanos , Indicadores y Reactivos , Masculino , Espectrometría de Masas , Péptidos/química , Próstata/química , Próstata/citología , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Biosíntesis de ProteínasRESUMEN
Enoxacin is a broad-spectrum quinolone-derivative antibiotic. In a rabbit model of keratitis caused by a Pseudomonas species, enoxacin (3 mg/mL) was as effective as gentamicin sulfate (3 mg/mL) and enoxacin (10 mg/mL) in reducing viable bacterial counts in corneas after 24 hours of hourly therapy with eye drops. Bacterial counts were reduced by about 5000-fold by enoxacin treatment when compared with placebo-treated controls. Penetration studies of topical enoxacin (3 mg/mL) showed that concentrations in cornea and aqueous humor reached levels above reported minimal inhibitory concentrations when an epithelial defect was present. Further investigation of enoxacin for treatment of ocular disease is warranted.
Asunto(s)
Antiinfecciosos/uso terapéutico , Úlcera de la Córnea/tratamiento farmacológico , Naftiridinas/uso terapéutico , Infecciones por Pseudomonas/tratamiento farmacológico , Animales , Antiinfecciosos/metabolismo , Humor Acuoso/metabolismo , Córnea/metabolismo , Úlcera de la Córnea/metabolismo , Úlcera de la Córnea/microbiología , Evaluación de Medicamentos , Enoxacino , Gentamicinas/uso terapéutico , Naftiridinas/metabolismo , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , ConejosRESUMEN
In this randomized, open-label, dose-comparative study, 18 investigators enrolled 466 patients with acute bronchitis. Patients were randomly assigned to receive either 600 mg of cefdinir once daily (QD) or 300 mg of cefdinir twice daily (BID) for 10 days. Both microbiologic and clinical efficacy were assessed at the test-of-cure visit, 7 to 14 days after therapy stopped. A total of 296 patients were classified as assessable at the test-of-cure visit (n = 150 QD, n = 146 BID). Eradication rates of baseline pathogens in these assessable patients were similar in both groups; the baseline pathogen eradication rate for assessable patients in the QD arm was 92%, and that in the BID arm was 93%. Clinical success (cure or improvement) in assessable patients was 91% and 93%, respectively. No difference was seen in the incidence of adverse events, in the incidence of diarrhea, or in the incidence of treatment withdrawals between the two groups. We conclude that cefdinir is effective and safe for the treatment of patients with acute bronchitis.
Asunto(s)
Bronquitis/tratamiento farmacológico , Cefalosporinas/uso terapéutico , Adulto , Cefdinir , Cefalosporinas/efectos adversos , Femenino , Humanos , MasculinoRESUMEN
Implant placement in the posterior maxilla often requires elevation of the sinus floor, which can be achieved through either the modified Caldwell-Luc or the crestal osteotome technique. The objectives of this study were to evaluate (a) the resistance to perforation of maxillary sinus membranes obtained from formaldehyde-fixed cadavers in vitro, (b) the frequency and extent of membrane perforations occurring after sinus floor elevation in cadavers using the crestal approach, and (c) the amount of membrane elevation (doming) that can be achieved using the crestal approach. Pretreatment of maxillary sinus membrane tissues with commonly used tissue softeners did not have a statistically significant effect on resistance to perforation. Maxillary sinus membranes were elevated 4 to 8 mm in formaldehyde-fixed cadavers using the osteotome technique; implants were placed. Of the 25 sites that received implants, only 6 showed perforations, as assessed by double-blind investigation after dissection of the lateral wall of the nose, allowing direct examination of the sinus cavity. Perforations were categorized as Class I (< or = 2 mm with exposure of the implant into the sinus cavity and loss of doming); Class II perforations (> or = 2 mm) were associated with proximity of the osteotomy site to the medial wall of the sinus or the presence of septae. These results indicated that the crestal osteotome approach compared favorably to the modified Caldwell-Luc technique as it relates to the frequency of maxillary sinus membrane perforations and the degree of achievable membrane elevation.
Asunto(s)
Seno Maxilar/cirugía , Procedimientos Quirúrgicos Preprotésicos Orales/métodos , Materiales Biocompatibles , Vidrio , Humanos , Membrana Mucosa/lesiones , Procedimientos Quirúrgicos Preprotésicos Orales/efectos adversos , Osteotomía/efectos adversos , Osteotomía/métodos , Distribución Aleatoria , Acondicionamiento de Tejidos Dentales , Heridas Penetrantes/etiología , Heridas Penetrantes/prevención & controlRESUMEN
The rate of scientific discovery can be accelerated through computation and visualization. This acceleration results from the synergy of expertise, computing tools, and hardware for enabling high-performance computation, information science, and visualization that is provided by a team of computation and visualization scientists collaborating in a peer-to-peer effort with the research scientists. In the context of this discussion, high performance refers to capabilities beyond the current state of the art in desktop computing. To be effective in this arena, a team comprising a critical mass of talent, parallel computing techniques, visualization algorithms, advanced visualization hardware, and a recurring investment is required to stay beyond the desktop capabilities. This article describes, through examples, how the Scientific Applications and Visualization Group (SAVG) at NIST has utilized high performance parallel computing and visualization to accelerate condensate modeling, (2) fluid flow in porous materials and in other complex geometries, (3) flows in suspensions, (4) x-ray absorption, (5) dielectric breakdown modeling, and (6) dendritic growth in alloys.
RESUMEN
Metagenomics uses gene expression patterns to understand the taxonomy and metabolic activities of microbial communities. Metaproteomics applies the same approach to community proteomes. Previously, we used a novel three-dimensional peptide separation method to identify over 2000 salivary proteins. This study used those data to carry out the first metaproteomic analysis of the human salivary microbiota. The metagenomic software MEGAN generated a phylogenetic tree, which was checked against the Human Oral Microbiome Database (HOMD). Pathway analyses were performed with the Clusters of Orthologous Groups and MetaCyc databases. Thirty-seven per cent of the peptides were identifiable only at the level of cellular organisms or bacteria. The rest were distributed among five bacterial phyla (61%), archea (0.5%), and viruses (0.8%); 29% were assignable at the genus level, and most belonged to Streptococcus (17%). Eleven per cent of all peptides could be assigned to species. Most taxa were represented in HOMD and they included well-known species such as periodontal pathogens. However, there also were 'exotic' species including aphid endosymbionts; plant, water, and soil bacteria; extremophiles; and archea. The pathway analysis indicated that peptides were linked to translation (37%), followed by glycolysis (19%), amino acid metabolism (8%), and energy production (8%). The taxonomic structure of the salivary metaproteome is very diverse but is dominated by streptococci. 'Exotic' species may actually represent close relatives that have not yet been sequenced. Salivary microbes appear to be actively engaged in protein synthesis, and the pathway analysis is consistent with the metabolism of salivary glycoproteins.
Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Proteoma/análisis , Saliva/microbiología , Sistemas de Transporte de Aminoácidos , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/metabolismo , Bases de Datos de Proteínas , Metabolismo Energético , Glucólisis , Humanos , Péptidos/análisis , Filogenia , Biosíntesis de Proteínas , Proteoma/genética , Proteínas y Péptidos Salivales/análisis , Espectrometría de Masas en TándemRESUMEN
The recF gene of Escherichia coli is known to encode an Mr-40,000 protein that is involved in DNA recombinationa nd postreplication DNA repair. To characterize the role of the recF gene product in these processes, the recF gene was cloned downstream of a tac promoter to facilitate overproduction of the recF gene product. The RecF protein was overproduced and purified to apparent homogeneity. N-terminal protein sequence analysis demonstrated that the purified protein had the sequence that was predicted from the DNA sequence of the recF gene, except that the predicted N-terminal Met was not present. The RecF protein bound to single-stranded oligonucleotides in filter binding and gel filtration assays. Maximal binding required 2 to 3 min of incubation at 37 degrees C; the binding reaction had a pH optimum of 7.0, did not require divalent cations, and was inhibited by NaCl concentrations of greater than 250 mM. The Kd of RecF protein binding to a 59-base single-stranded oligonucleotide was on the order of 1.3 X 10(-7) M, and the reaction did not show cooperativity. Experiments measuring the binding to various DNA substrates and competition binding experiments with different DNA molecules demonstrated that RecF protein binds preferentially to single-stranded, linear DNA molecules.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Genes Bacterianos , Datos de Secuencia Molecular , Peso Molecular , Sondas de Oligonucleótidos , Plásmidos , Mapeo Restrictivo , Especificidad por SustratoRESUMEN
An approach to genetic identification using biallelic single-nucleotide polymorphism (SNP) genetic markers is described in which the three possible genotypes, AA, Aa, or aa, where "A" and "a" represent the two SNP alleles, are assigned a ternary (base 3) digit of 0, 1, or 2, respectively. Genotyping an individual over a panel of separate SNP markers produces a composite ternary genetic code that can be converted to an easily stored, decimal (base 10) genetic identification number. The unambiguous identification of 11 individuals is demonstrated using ternary genetic codes generated from MALDI-TOF mass spectrometric genotyping data from 7 different SNP markers.
Asunto(s)
Genotipo , Polimorfismo Genético/genética , Alelos , ADN/análisis , ADN/genética , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
An approach is described for predicting peptide nucleic acid (PNA):DNA duplex stability from base sequence by approximating the total free energy of dissociation, delta G degree tot, for these duplexes as the sum of five parameters: (a) a nearest-neighbor interaction summation term, sigma Nj delta G degree j; (b) an initiation term, delta G degree i; (c) a dangling-end stabilization term, delta G degree e; (d) a PNA:DNA stabilization term per nearest-neighbor interaction, delta G degree pna; and (e) an ionic strength term, delta G degree Na. Parameters (a) and (b) are approximated using previously determined values for DNA:DNA duplexes, whereas parameters (c), (d), and (e) are empirically determined. These terms are used to calculated delta G degree tot, which is used in conjunction with a transition enthalpy (delta H degree) value, also approximated from nearest-neighbor values previously derived for DNA:DNA duplexes, to calculate a melting temperature (Tm) for the PNA:DNA duplex. Predicted Tm values calculated by this approach agreed fairly well with measured values for 11 different PNA:DNA duplexes, as well as with literature values. The approach also accurately models ionic strength effects.
Asunto(s)
ADN/química , Oligonucleótidos/química , Secuencia de Bases , TermodinámicaRESUMEN
A case of IP in a 16-year-old girl has been presented. This patient manifested classic ectodermal and mesodermal anomalies. We present this case to illustrate a rare etiologic factor in the development of dentofacial deformities that can be treated in the conventional manner.
Asunto(s)
Atención Dental para la Persona con Discapacidad , Huesos Faciales/anomalías , Incontinencia Pigmentaria , Anomalías Dentarias/terapia , Adolescente , Anodoncia/cirugía , Implantación de Cuchilla (Odontología) , Hilos Ortopédicos , Asimetría Facial/cirugía , Huesos Faciales/cirugía , Femenino , Humanos , Inmovilización , Osteotomía , Trastornos de la Articulación Temporomandibular/cirugíaRESUMEN
The Tn552 transposase, a member of the DDE superfamily of transposase and retroviral integrase proteins, has been expressed in soluble form. The purified protein performs concerted strand transfer in vitro, efficiently pairing two preprocessed transposon ends and inserting them into target DNA. For maximum efficiency, both participating DNA ends must contain the two adjacent transposase-binding sites that are the normal constituents of the Tn552 termini. As is the case with transposition in vivo, the insertions recovered from the reaction in vitro are flanked by repeats of a short target sequence, most frequently 6 bp. The reaction has stringent requirements for a divalent metal ion. Concerted strand transfer is most efficient with Mg2+. Although it stimulates strand transfer overall, Mn2+ promotes uncoupled, single-ended events at the expense of concerted insertions. The simplicity and efficiency of the Tn552 transposition system make it an attractive subject for structural and biochemical studies and a potentially useful genetic tool.
Asunto(s)
ADN/metabolismo , Transposasas/metabolismo , Secuencia de Bases , Catálisis , Clonación Molecular , Elementos Transponibles de ADN , Magnesio/metabolismo , Manganeso/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , Desnaturalización Proteica , Secuencias Repetitivas de Ácidos Nucleicos , SolubilidadRESUMEN
Campylobacter jejuni is the most common cause of food-borne illnesses in the United States. Despite the fact that the entire nucleotide sequence of its genome has recently become available, its mechanisms of pathogenicity are poorly understood. This is in part due to the lack of an efficient mutagenesis system. Here we describe an in vitro transposon mutagenesis system based on the Staphylococcus aureus transposable element Tn552 that allows the efficient generation of insertion mutants of C. jejuni. Insertions occur randomly and throughout the entire bacterial genome. We have tested this system in the isolation of nonmotile mutants of C. jejuni. Demonstrating the utility of the system, six nonmotile mutants from a total of nine exhibited insertions in genes known to be associated with motility. An additional mutant had an inactivating insertion in sigma 54, implicating this transcription factor in flagellum regulation. The availability of this efficient system will greatly facilitate the study of the mechanisms of pathogenesis of this important pathogen.
Asunto(s)
Campylobacter jejuni/genética , Elementos Transponibles de ADN , Secuencia de Bases , Datos de Secuencia Molecular , MutagénesisRESUMEN
Six hundred ninety patients were enrolled in a multicenter, randomized, double-blind trial comparing the efficacy and safety of cefdinir with those of cefaclor in the treatment of community-acquired pneumonia. Patients received either 10 days of treatment with cefdinir (n = 347) at 300 mg twice daily or 10 days of treatment with cefaclor (n = 343) at 500 mg three times daily. Microbiological assessments were performed on sputum specimens obtained at admission and at the two posttherapy visits, if available. Respiratory tract pathogens were isolated from 538 (78%) of 690 patient admission sputum specimens, with the predominant pathogens being Haemophilus parainfluenzae, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus. The microbiological eradication rates at the test-of-cure visit were 92% (238 of 260 pathogens) and 93% (245 of 264 pathogens) for the evaluable patients treated with cefdinir and cefaclor, respectively. A satisfactory clinical response (cure plus improvement) was achieved in 89% (166 of 187) and 86% (160 of 186) of the evaluable patients treated with cefdinir and cefaclor, respectively. Except for the incidence of diarrhea, adverse event rates while on treatment were equivalent between the two treatment groups. Diarrhea incidence during therapy was higher for patients treated with cefdinir (13.7%) than for patients treated with cefaclor (5.3%). These results indicate that cefdinir is effective and safe in the treatment of patients with pneumonia.
Asunto(s)
Cefaclor/uso terapéutico , Cefalosporinas/uso terapéutico , Infecciones Comunitarias Adquiridas/tratamiento farmacológico , Neumonía Bacteriana/tratamiento farmacológico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Cefaclor/efectos adversos , Cefdinir , Cefalosporinas/efectos adversos , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Resultado del Tratamiento , Estados UnidosRESUMEN
An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.