RESUMEN
MOTIVATION: Estimating the effects of interventions on patient outcome is one of the key aspects of personalized medicine. Their inference is often challenged by the fact that the training data comprises only the outcome for the administered treatment, and not for alternative treatments (the so-called counterfactual outcomes). Several methods were suggested for this scenario based on observational data, i.e. data where the intervention was not applied randomly, for both continuous and binary outcome variables. However, patient outcome is often recorded in terms of time-to-event data, comprising right-censored event times if an event does not occur within the observation period. Albeit their enormous importance, time-to-event data are rarely used for treatment optimization. We suggest an approach named BITES (Balanced Individual Treatment Effect for Survival data), which combines a treatment-specific semi-parametric Cox loss with a treatment-balanced deep neural network; i.e. we regularize differences between treated and non-treated patients using Integral Probability Metrics (IPM). RESULTS: We show in simulation studies that this approach outperforms the state of the art. Furthermore, we demonstrate in an application to a cohort of breast cancer patients that hormone treatment can be optimized based on six routine parameters. We successfully validated this finding in an independent cohort. AVAILABILITY AND IMPLEMENTATION: We provide BITES as an easy-to-use python implementation including scheduled hyper-parameter optimization (https://github.com/sschrod/BITES). The data underlying this article are available in the CRAN repository at https://rdrr.io/cran/survival/man/gbsg.html and https://rdrr.io/cran/survival/man/rotterdam.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Redes Neurales de la Computación , Programas Informáticos , Simulación por Computador , Humanos , Medicina de Precisión , ProbabilidadRESUMEN
MOTIVATION: In biomedicine, every molecular measurement is relative to a reference point, like a fixed aliquot of RNA extracted from a tissue, a defined number of blood cells, or a defined volume of biofluid. Reference points are often chosen for practical reasons. For example, we might want to assess the metabolome of a diseased organ but can only measure metabolites in blood or urine. In this case, the observable data only indirectly reflects the disease state. The statistical implications of these discrepancies in reference points have not yet been discussed. RESULTS: Here, we show that reference point discrepancies compromise the performance of regression models like the LASSO. As an alternative, we suggest zero-sum regression for a reference point insensitive analysis. We show that zero-sum regression is superior to the LASSO in case of a poor choice of reference point both in simulations and in an application that integrates intestinal microbiome analysis with metabolomics. Moreover, we describe a novel coordinate descent based algorithm to fit zero-sum elastic nets. AVAILABILITY AND IMPLEMENTATION: The R-package "zeroSum" can be downloaded at https://github.com/rehbergT/zeroSum Moreover, we provide all R-scripts and data used to produce the results of this manuscript as Supplementary Material CONTACT: Michael.Altenbuchinger@ukr.de, Thorsten.Rehberg@ukr.de and Rainer.Spang@ukr.deSupplementary information: Supplementary material is available at Bioinformatics online.
Asunto(s)
Bacterias/metabolismo , Biología Computacional/métodos , Metabolómica , Programas Informáticos , Algoritmos , Bacterias/genética , Simulación por Computador , Microbioma Gastrointestinal/genética , Regulación Bacteriana de la Expresión Génica , HumanosRESUMEN
MOTIVATION: Molecular signatures for treatment recommendations are well researched. Still it is challenging to apply them to data generated by different protocols or technical platforms. RESULTS: We analyzed paired data for the same tumors (Burkitt lymphoma, diffuse large B-cell lymphoma) and features that had been generated by different experimental protocols and analytical platforms including the nanoString nCounter and Affymetrix Gene Chip transcriptomics as well as the SWATH and SRM proteomics platforms. A statistical model that assumes independent sample and feature effects accounted for 69-94% of technical variability. We analyzed how variability is propagated through linear signatures possibly affecting predictions and treatment recommendations. Linear signatures with feature weights adding to zero were substantially more robust than unbalanced signatures. They yielded consistent predictions across data from different platforms, both for transcriptomics and proteomics data. Similarly stable were their predictions across data from fresh frozen and matching formalin-fixed paraffin-embedded human tumor tissue. AVAILABILITY AND IMPLEMENTATION: The R-package 'zeroSum' can be downloaded at https://github.com/rehbergT/zeroSum . Complete data and R codes necessary to reproduce all our results can be received from the authors upon request. CONTACT: rainer.spang@ur.de.
Asunto(s)
Linfoma de Burkitt/genética , Biología Computacional/métodos , Linfoma de Células B Grandes Difuso/genética , Proteoma , Programas Informáticos , Conservación de Tejido , Transcriptoma , Algoritmos , Linfoma de Burkitt/metabolismo , Formaldehído , Congelación , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Modelos Estadísticos , Adhesión en ParafinaRESUMEN
Binary polymorphisms associated with the non-recombining region of the human Y chromosome (NRY) preserve the paternal genetic legacy of our species that has persisted to the present, permitting inference of human evolution, population affinity and demographic history. We used denaturing high-performance liquid chromatography (DHPLC; ref. 2) to identify 160 of the 166 bi-allelic and 1 tri-allelic site that formed a parsimonious genealogy of 116 haplotypes, several of which display distinct population affinities based on the analysis of 1062 globally representative individuals. A minority of contemporary East Africans and Khoisan represent the descendants of the most ancestral patrilineages of anatomically modern humans that left Africa between 35,000 and 89,000 years ago.
Asunto(s)
Etnicidad/genética , Evolución Molecular , Hominidae/genética , Filogenia , Cromosoma Y/genética , África , Animales , Cromatografía Líquida de Alta Presión , Haplotipos/genética , Humanos , Masculino , Modelos Genéticos , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.
Asunto(s)
Arabidopsis/genética , Marcadores Genéticos/genética , Genoma de Planta , Ascomicetos/crecimiento & desarrollo , Mapeo Cromosómico , ADN de Plantas/genética , Genes de Plantas/genética , Predisposición Genética a la Enfermedad , Genotipo , Análisis de Secuencia por Matrices de Oligonucleótidos , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Polimorfismo GenéticoRESUMEN
Milk production in dairy cows has dramatically increased over the past few decades. The selection for higher milk yield affects the partitioning of available nutrients, with more energy being allocated to milk synthesis and less to physiological processes essential to fertility and fitness. In this study, the abundance of numerous milk metabolites in early and late lactation was systematically investigated, with an emphasis on metabolites related to energy metabolism. The aim of the study was the identification and correlation of milk constituents to the metabolic status of the cows. To investigate the influence of lactation stage on physiological and metabolic variables, 2 breeds of different productivity were selected for investigation by high-resolution nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. We could reliably quantify 44 different milk metabolites. The results show that biomarkers such as acetone and beta-hydroxybutyrate are clearly correlated to the metabolic status of the individual cows during early lactation. Based on these data, the selection of cows that cope well with the metabolic stress of early lactation should become an option.
Asunto(s)
Adaptación Fisiológica , Bovinos/fisiología , Metabolismo Energético/fisiología , Lactancia/metabolismo , Leche/química , Ácido 3-Hidroxibutírico/análisis , Acetona/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Femenino , Cromatografía de Gases y Espectrometría de Masas , Espectroscopía de Resonancia MagnéticaRESUMEN
A genetic perspective of human history in Europe was derived from 22 binary markers of the nonrecombining Y chromosome (NRY). Ten lineages account for >95% of the 1007 European Y chromosomes studied. Geographic distribution and age estimates of alleles are compatible with two Paleolithic and one Neolithic migratory episode that have contributed to the modern European gene pool. A significant correlation between the NRY haplotype data and principal components based on 95 protein markers was observed, indicating the effectiveness of NRY binary polymorphisms in the characterization of human population composition and history.
Asunto(s)
Pool de Genes , Genética de Población , Cromosoma Y , Alelos , Antropología Física , Clima , ADN Mitocondrial/genética , Emigración e Inmigración , Europa (Continente) , Femenino , Marcadores Genéticos , Historia Antigua , Humanos , Masculino , Medio OrienteRESUMEN
Allelic discrimination of single nucleotide polymorphisms (SNPs) and, particularly, determination of the phase of multiple variations are of utmost importance in genetics. The physicochemical separation of alleles by completely denaturing ion-pair reversed-phase high-performance liquid chromatography and their on-line sequence determination by electrospray ionization mass spectrometry is demonstrated. Simultaneous genotyping of two and three simple sequence polymorphisms contained within 73-114 bp was accomplished with low femtomolar amounts of unpurified amplicons from polymerase chain reaction. Determination of allelic composition is enabled by the high accuracy (better than 0.019%) of intact mass measurements or by comparative sequencing using gas-phase fragmentation and tandem mass spectrometry in combination with fully automated, computer-aided data interpretation.
Asunto(s)
Cromatografía Liquida/métodos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Genotipo , Haplotipos , Humanos , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Lugares Marcados de SecuenciaRESUMEN
Intestinal dysbiosis has been associated with acute gastrointestinal GvHD and poor outcome following allogeneic stem cell transplantation (ASCT). To assess the effect of a switch in 2012 from ciprofloxacin/metronidazole to rifaximin for gut decontamination on intestinal microbiota composition and ASCT outcome, we retrospectively analyzed 394 patients receiving ASCT from September 2008 through June 2015. In 131 and 90 patients, respectively, urinary 3-indoxyl sulfate levels and intestinal enterococcal load were measured before conditioning and weekly within the first 28 days after ASCT. The use of rifaximin correlated with lower enterococcal positivity (6.9 vs 21.9%, P=0.05) and higher urinary 3-indoxyl sulfate concentrations (10.5 vs 4.6 µmoL/mmoL crea, P<0.001) after ASCT. Patients on rifaximin showed lower 1-year transplant-related mortality (P=0.04) and higher overall survival (P=0.008). Treatment of infectious complications with systemic antibiotics did not abrogate the beneficial effects of rifaximin on intestinal microbiota composition in the early course of ASCT and outcome. The data underscore the importance of maintaining a diverse population of symbiotic and mutualistic bacteria in the gut on ASCT outcome.
Asunto(s)
Microbioma Gastrointestinal/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas/métodos , Rifamicinas/administración & dosificación , Adulto , Enterococcus/efectos de los fármacos , Femenino , Enfermedades Gastrointestinales/prevención & control , Enfermedad Injerto contra Huésped/microbiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/mortalidad , Humanos , Indicán/análisis , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Rifamicinas/farmacología , Rifaximina , Análisis de Supervivencia , Trasplante HomólogoRESUMEN
We previously used gel shift assays, Southwestern blots, and UV cross-linking to identify four proteins that bind to the 203-bp 5'-flanking region (-194/ +9) of the rabbit uteroglobin gene. Here we report cloning, by recognition site screening, the cDNAs for two of the uteroglobin promoter-binding proteins (95 kDa and 113 kDa). Their presumptive nucleotide-binding motifs share 61% identity with the SWI2/SNF2 helicase superfamily, and each protein has the novel C3HC4 (RING) zinc-finger signature near its C terminus. RUSH-1 alpha, the 113-kDa protein, is the rabbit homolog of human HIP116, a protein that binds to the human immunodeficiency virus-1 promoter. RUSH-1 beta is a 95-kDa truncated version of RUSH-1 alpha that results from alternative splicing of a 57-bp exon as confirmed by genomic cloning. Northern analysis showed mRNA expression (5.2 kb) was induced by progesterone +/- PRL and antagonized by estrogen. However, because the two proteins result from alternative splicing of a 57-bp exon, the small difference in their mRNA sizes could not be detected by Northern analysis. Therefore, competitive RT-PCR and HPLC were used to quantify differences in the ratios of their mRNAs. Progesterone +/- PRL treatment increased (P < 0.005) the ratio of message for RUSH-1 alpha compared with RUSH-1 beta. Western analysis showed the RUSH-1 alpha protein is increased in response to progesterone +/- PRL and decreased in response to estrogen. The antiserum used for immunoblotting specifically supershifts uteroglobin promoter-protein complexes in gel shift experiments. Because RUSH-1 alpha and beta messages were detected in lung, liver, and HRE-H9 cells, these proteins may regulate genes in numerous cell types.
Asunto(s)
Empalme Alternativo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares , Esteroides/metabolismo , Uteroglobina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión/métodos , Clonación Molecular , ADN Helicasas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/efectos de los fármacos , Endometrio/metabolismo , Estro/metabolismo , Femenino , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Progesterona/metabolismo , Progesterona/farmacología , Prolactina/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Conejos , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , Uteroglobina/metabolismoRESUMEN
Vitamin D has emerged as a central player in the immune system, with its deficiency being implicated in the pathogenesis of several autoimmune diseases, including chronic GvHD. This is a retrospective cohort analysis of 166 patients, who underwent allogeneic hematopoietic stem cell transplantation (HSCT) at the Karolinska University Hospital, evaluating GvHD, graft failure, infectious complications and survival after HSCT in relation to pre-transplantation vitamin D levels. Most of the patients were deficient in vitamin D before HSCT (median 42 nmol/L). In multivariate analysis, vitamin D level before HSCT was identified as a significant independent risk factor for development of cGvHD. The increased incidence of cGvHD was not coupled to better disease-free survival; instead there was a trend towards lower overall survival in the vitamin D-deficient patients. In addition, we found a significant correlation between vitamin D deficiency and incidence of CMV disease, with no case of CMV disease occurring in patients with sufficient levels of vitamin D before HSCT. Our results support a role of vitamin D in immune tolerance following HSCT. These findings could be highly relevant for the care of HSCT patients, and prospective, randomized studies on the effect of vitamin D supplementation are therefore needed.
Asunto(s)
Infecciones por Citomegalovirus/epidemiología , Citomegalovirus , Enfermedad Injerto contra Huésped/epidemiología , Trasplante de Células Madre Hematopoyéticas , Neoplasias/terapia , Deficiencia de Vitamina D/epidemiología , Adulto , Aloinjertos , Enfermedad Crónica , Infecciones por Citomegalovirus/etiología , Infecciones por Citomegalovirus/terapia , Femenino , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/terapia , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Neoplasias/epidemiología , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/terapiaRESUMEN
Denaturing high-performance liquid chromatography (DHPLC) compares two or more chromosomes as a mixture of denatured and reannealed PCR amplicons, revealing the presence of a mutation by the differential retention of homo- and heteroduplex DNA on reversed-phase chromatography supports under partial denaturation. Temperature determines sensitivity, and its optimum can be predicted by computation. Single-nucleotide substitutions, deletions, and insertions have been detected successfully by on-line UV or fluorescence monitoring within 2-3 minutes in unpurified amplicons as large as 1.5 Kb. Sensitivity and specificity of DHPLC consistently exceed 96%. These features and its low cost make DHPLC one of the most powerful tools for the re-sequencing of the human and other genomes. Aside from its application to the mutational analysis of candidate genes, DHPLC has proven instrumental in elucidating human evolution and in the mapping of genes. Employing completely denaturing conditions, the utility of DHPLC has been extended to the genotyping of known polymorphisms by utilizing the ability of poly(styrene-divinylbenzene) to resolve single-stranded DNA molecules of identical size that differ in a single base. Under completely denaturing conditions, it is thus possible to resolve all possible base substitutions with the single exception of C-->G transversions. Improvements in throughput became feasible with the recent introduction of monolithic poly(styrene-divinylbenzene) capillaries that lend themselves to the fabrication of arrays connected to a multi-color laser induced fluorescence scanner or a mass spectrometer.
Asunto(s)
Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN/métodos , Secuencia de Bases , Mutación , Polimorfismo GenéticoRESUMEN
An assessment of 28 pertinent binary genetic markers on the non-recombining portion of the Y chromosome (NRY) in New Zealand Maori and other relevant populations has revealed a diverse genetic paternal heritage of extant Maori. A maximum parsimony phylogeny was constructed in which nine of the 25 possible binary haplotypes were observed. Although approximately 40% of the samples have haplotypes of unequivocal European origin, an equivalent number of samples have a single binary haplotype that is also observed in Indonesia and New Guinea, indicative of common indigenous Melanesian ancestry. The balance of the lineages has either typical East Asian signatures or alternative compositions consistent with their affinity to Melanesia or New Guinea. Molecular analysis of mtDNA variation confirms the presence of a single predominant characteristic Southeast Asian (9-bp deletion in the Region V) lineage. The Y-chromosome results support a pattern of complex interrelationships between Southeast Asia, Melanesia, and Polynesia, in contrast to mtDNA and linguistic data, which uphold a rapid and homogeneous Austronesian expansion. The Y-chromosome data highlight a distinctive gender-modulated pattern of differential gene flow in the history of Polynesia.
Asunto(s)
ADN Mitocondrial/genética , Etnicidad/genética , Haplotipos/genética , Filogenia , Población Blanca/genética , Cromosoma Y/genética , Cromatografía Líquida de Alta Presión , Femenino , Frecuencia de los Genes/genética , Variación Genética/genética , Humanos , Lingüística , Masculino , Repeticiones de Microsatélite/genética , Desnaturalización de Ácido Nucleico , Islas del Pacífico , Reacción en Cadena de la Polimerasa , Polimorfismo Genético/genéticaRESUMEN
Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and the ion-pair reverse-phase (IP-RP)-HPLC product purification and detection system were developed to facilitate the isolation and proportional quantification of alternatively spliced RUSH mRNAs. RUSH isoforms result from alternative splicing of a 57-bp exon and encode SNF/SWI-related proteins that bind to the uteroglobin promoter. QRT-PCR was performed using total RNA, and a pair of primers designed to flank the 57-bp exon. When more than one splice variant was expressed, IP-RP-HPLC identified the specific homoduplex products, as well as the heteroduplexes formed as a consequence of partial sequence complementarity between the products. Data analysis included the correct re-allocation of heteroduplex components to achieve accurate quantitation of changes in the relative levels of RUSH message isoforms. The preferential expression of the RUSH-1alpha isoform by all the tissues except estrous uterine endometrium and lactating mammary gland indicates RUSH pre-mRNAs are alternatively spliced in a tissue-specific manner. A 61-fold difference in the relative rate of RUSH pre-mRNA splicing is indicated by the difference in the ratios of RUSH mRNA isoforms from uterine endometrium and testis. Clearly, QRT-PCR and IP-RP-HPLC are powerful and versatile tools for the detection and quantitation of mRNA splice variants.
Asunto(s)
Empalme Alternativo , Proteínas de Unión al ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Animales , Cromatografía Líquida de Alta Presión/métodos , Regulación del Desarrollo de la Expresión Génica , ARN Mensajero/genética , Conejos , Distribución Tisular , Factores de Transcripción/genética , Uteroglobina/genéticaRESUMEN
OBJECTIVE: To determine the qualitative and quantitative expression of alpha and gamma sodium pump subunits in whole kidney and nephron segment RNA from Sprague Dawley rats, spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. DESIGN: A novel reverse transcription polymerase chain reaction technique was devised which provides accurate and precise measurement of the number of molecules of specific transcript abundance, a measurement of gene expression. This allows the quantitative comparison of multiple samples across multiple subjects and, since the estimates are accurate rather than relative, can also be used to make quantitative comparisons across expressed genes, such as isoforms and subunits of the heterotrimeric renal sodium pump. METHODS: We examined which catalytic isoforms were expressed and then quantified transcript abundance in whole kidney and convoluted and straight segments of the proximal tubule. RESULTS: Alpha 1 and gamma transcripts, but not alpha 2, alpha 3 or alpha 4 isoforms, were consistently observed in nephron segments. Levels of alpha 1 were lower in kidney RNA from 15-16-week-old SHR than in WKY rats of the same age (P = 0.001), but were not different between SHR and WKY in 4-5-week-old animals. No significant difference was observed in gamma subunit abundance in kidney RNA from 4-5-week-old animals; however, at 15-16 weeks, the expression in SHR was one-third that in WKY rats (P = 0.003). In proximal convoluted tubules from 4-5-week-old animals, the level of alpha 1 RNA expression was lower (P = 0.03) in SHR than in WKY rats. In addition, levels of alpha 1 in proximal straight tubule from the 4-5-week-old SHR were also lower than in WKY rats (P = 0.02). This difference was even greater in 15-16-week-old animals: in SHR, alpha 1 expression was less than 20% of the level of expression in WKY rats (P = 0.0003). Expression of the gamma subunit exhibited a similar pattern of downregulation in SHR. In RNA from proximal convoluted tubules and proximal straight tubules from both 4-5- and 15-16-week-old animals, expression of the gamma subunit was demonstrated to be significantly lower in SHR than in WKY rats. CONCLUSION: The results indicate a coordinate reduction in the abundance of sodium pump alpha and gamma subunits in the proximal tubules of SHR, which occurs early during the development of hypertension.
Asunto(s)
Hipertensión/metabolismo , Nefronas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Expresión Génica , Hipertensión/genética , Riñón/metabolismo , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Genetic maps based on biallelic single-nucleotide polymorphisms amenable to microarray-based genotyping have significantly accelerated the mapping of mono- and multigenic traits in model organisms such as Saccharomyces cerevisiae and Arabidopsis thaliana. This advance needs to be matched by highly accurate, inexpensive and robust methodology for fine-structure mapping of the candidate region(s) and the eventual identification of the causative mutation(s). To establish the usefulness of denaturing high-performance liquid chromatography (DHPLC) for those purposes, we have amplified 476 fragments from two A. thaliana ecotypes with an average length of 563 bp covering various candidate regions on chromosomes 1, 2 and 4. Parallel analysis by DHPLC and dye terminator sequencing showed that DHPLC detected 165 out of 166 polymorphic fragments with only four false positives, amounting to a sensitivity, specificity and accuracy of 99.4%, 98.7% and 99%, respectively. It proved beneficial to analyze the fragments not only at the highest but also at the lower temperatures recommended by the algorithm freely available at http:¿insertion.stanford.edu/melt.html.
Asunto(s)
Arabidopsis/genética , Cromatografía Líquida de Alta Presión/métodos , Análisis Mutacional de ADN/métodos , Variación Genética/genética , Desnaturalización de Ácido Nucleico , Algoritmos , Alelos , Arabidopsis/clasificación , Cromosomas/genética , Cromosomas Artificiales Bacterianos/genética , Cartilla de ADN , Genes de Plantas/genética , Mutación/genética , Ácidos Nucleicos Heterodúplex/análisis , Ácidos Nucleicos Heterodúplex/genética , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple/genética , Sensibilidad y Especificidad , TemperaturaRESUMEN
Competitive reverse-transcription PCR (RT-PCR) techniques for quantification of gene expression employ titrations in which the products of multiple PCRs must be separated, analyzed and quantified to compute gene expression in a single sample. We have employed a novel, ion-pair reversed-phase HPLC (IP-RP-HPLC) system to analyze and quantify RT-PCRs performed with mutant RNA internal standards. PCR products could be separated and quantified in 6 minutes per reaction using the absorbance signal from an on-line UV detector. Crude PCR products can be analyzed without further processing and without the addition of radioactive or fluorescent markers to reactions. Analysis of titration regression and slope values approached mathematical ideals indicating that amplification of native and competitor RNA occurred with equal efficiency. Further, serial dilution of input RNA over three orders of magnitude did not affect the calculated level of gene expression or the slope of the titration. IP-RP-HPLC appears to offer important advantages to quantitative measurements of gene expression. These include rapid sample analysis and column re-equilibration, reduced sample handling and opportunity for introduction of quantification error, avoidance of fluorescent or radioactive tracers, high detector sensitivity and linearity and excellent quantitative reliability.
Asunto(s)
Cromatografía Líquida de Alta Presión , Expresión Génica/genética , Reacción en Cadena de la Polimerasa , Angiotensinógeno/genética , Animales , Secuencia de Bases , Encéfalo/metabolismo , Cartilla de ADN/química , Electroforesis en Gel de Agar , Isoenzimas/química , Isoenzimas/genética , Riñón/metabolismo , Modelos Lineales , Datos de Secuencia Molecular , ARN/análisis , ARN/metabolismo , Ratas , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificaciónRESUMEN
Denaturing high-performance liquid chromatography (DHPLC) is a sensitive, robust, and operationally inexpensive method for the detection of single-base substitutions and small deletions and insertions. To increase sample throughout, we have developed a multiplexing strategy using fluorophores to distinguish different PCR products. The system combines recent advances in the synthesis of monolithic poly(styrene-divinylbenzene) capillary columns with four-color confocal argon ion laser-induced fluorescence detection. Depending on the change in retention caused by the fluorophores, adjustments in the analysis temperature may be required to ensure the maximum mutation detection sensitivity.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Análisis Mutacional de ADN/métodos , Electroforesis Capilar/métodos , Fluorometría/instrumentación , Argón , Disparidad de Par Base , Cromatografía Líquida de Alta Presión/instrumentación , Análisis Mutacional de ADN/instrumentación , Electroforesis Capilar/instrumentación , Diseño de Equipo , Colorantes Fluorescentes/análisis , Análisis Heterodúplex , Humanos , Rayos Láser , Masculino , Desnaturalización de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/análisis , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Temperatura , Cromosoma Y/química , Cromosoma Y/genéticaRESUMEN
Ion-pair reversed-phase high-performance liquid chromatography on alkylated nonporous polystyrene-divinylbenzene particles with a mean diameter of 2.1 microns was used to analyze PCR products according to their chain length within a few minutes. The simple and reliable procedure allows the simultaneous separation and isolation of DNA fragments differing in chain length by 1%-5% up to a size of 500 base pairs with recovery rates exceeding 97%. A greater than 70-fold increase in sensitivity could be achieved through the use of a fluorescein-labeled primer, which allowed the determination of a 127-bp hepatitis C virus cDNA/PCR product with a lower mass detection limit of 2 fmol. Calibration curves showed excellent linearity over a range of at least 4 magnitudes. Finally, the stationary phase allowed the routine analysis of hundreds of PCR products with high reproducibility of both retention times and peak areas.