RESUMEN
Presentamos el caso de un paciente pediátrico que acudió a urgencias con dolor severo en la extremidad inferior derecha causado por la picadura de un escorpión. Ante la ausencia de respuesta a los analgésicos administrados se optó por realizar un bloqueo poplíteo ecoguiado, lo que consiguió una analgesia completa y permitió el manejo ambulatorio del paciente, sin referir efectos adversos. Las familias de escorpiones presentes en nuestro país no suponen un riesgo vital para el ser humano, pero su picadura produce una reacción local con dolor autolimitado a unas 24-48h que puede ser severo. El manejo prioritario es realizar una correcta analgesia. Las técnicas anestésicas regionales son de utilidad para el control del dolor agudo y pueden representar una colaboración eficaz entre los servicios de anestesiología y urgencias.(AU)
We report the case of a paediatric patient who presented at the Emergency Department with severe pain in the right lower extremity caused by a scorpion sting. Analgesics were ineffective, so we decided to perform an ultrasound-guided popliteal block. This, which achieved complete analgesia and allowed the patient to be followed up in the outpatient department, with no adverse effects. The sting of the species of scorpion found in Spain is not dangerous to human life; however, it causes self-limiting localised pain that lasts for 24-48hours, and can be severe. The first-line treatment is effective analgesia. Regional anaesthesia techniques are useful in the control of acute pain, and are an example of effective collaboration between the Anaesthesiology and Emergency services.(AU)
Asunto(s)
Humanos , Masculino , Niño , Nervio Ciático , Picaduras de Escorpión , Analgésicos , Urgencias Médicas , Dolor Agudo , Anestesia de Conducción , Pediatría , Pacientes Internos , Examen Físico , Anestesiología , UltrasonografíaRESUMEN
We report the case of a paediatric patient who presented at the Emergency Department with severe pain in the right lower extremity caused by a scorpion sting. Analgesics were ineffective, so we decided to perform an ultrasound-guided popliteal block, which provided complete analgesia and allowed the patient to be followed up in the outpatient department, with no adverse effects. The sting of the species of scorpion found in Spain is not dangerous to human life; however, it causes self-limiting localised pain that lasts for 24-48h, and can be severe. The first-line treatment is effective analgesia. Regional anaesthesia techniques are useful in the control of acute pain, and are an example of effective collaboration between the Anaesthesiology and Emergency services.
Asunto(s)
Dolor Agudo , Bloqueo Nervioso , Humanos , Niño , Animales , Escorpiones , Bloqueo Nervioso/métodos , Servicio de Urgencia en Hospital , Ultrasonografía Intervencional/métodosRESUMEN
A simple thermal annealing at 150 °C followed by exposure to air ambient conditions in epitaxial ZnO thin films produces a photoconductivity enhancement and a reduction of the energy gap. The first effect is related to a release of carriers from bulk traps while the second is caused by a gradual adsorption of species on the film surface which increases the band bending, as x-ray photoemission spectroscopy (XPS) shows. An observed drift of the photoconductivity and the energy gap over the days is connected to this adsorption kinetics. These findings have a potential application in ZnO based optoelectronic devices.
RESUMEN
The rapidly rising demand for therapeutic grade DNA molecules requires associated improvements in encapsulation and delivery technologies. One of the challenges for the efficient intracellular delivery of therapeutic biomolecules after their cell internalization by endocytosis is to manipulate the non-productive trafficking from endosomes to lysosomes, where degradation may occur. The combination of the endosomal acidity with the endosomolytic capability of the nanocarrier can increase the intracellular delivery of many drugs, genes and proteins, which, therefore, might enhance their therapeutic efficacy. Among the suitable compounds, the gelification properties of gelatin as well as the strong dependence of gelatin ionization with pH makes this compound an interesting candidate to be used to the effective intracellular delivery of active biomacromolecules. In the present work, gelatin (either high or low gel strength) and protamine sulfate has been selected to form particles by interaction of oppositely charged compounds. Particles in the absence of DNA (binary system) and in the presence of DNA (ternary system) have been prepared. The physicochemical characterization (particle size, polydispersity index and degree of DNA entrapment) have been evaluated. Cytotoxicity experiments have shown that the isolated systems and the resulting gelatin-based nanoparticles are essentially non-toxic. The pH-dependent hemolysis assay and the response of the nanoparticles co-incubated in buffers at defined pHs that mimic extracellular, early endosomal and late endo-lysosomal environments demonstrated that the nanoparticles tend to destabilize and DNA can be successfully released. It was found that, in addition to the imposed compositions, the gel strength of gelatin is a controlling parameter of the final properties of these nanoparticles. The results indicate that these gelatin-based nanoparticles have excellent properties as highly potent and non-toxic intracellular delivery systems, rendering them promising DNA vehicles to be used as non-viral gene delivery systems.
Asunto(s)
Materiales Biocompatibles , ADN/administración & dosificación , Gelatina/química , Nanopartículas/química , Células 3T3 , Animales , Portadores de Fármacos , Células HeLa , Hemólisis , Humanos , Ratones , Protaminas/química , RatasRESUMEN
Schizophrenia (SZ) and psychotic bipolar disorder (PBP) are disabling psychiatric illnesses with complex and unclear etiologies. Electroencephalogram (EEG) oscillatory abnormalities in SZ and PBP probands are heritable and expressed in their relatives, but the neurobiology and genetic factors mediating these abnormalities in the psychosis dimension of either disorder are less explored. We examined the polygenic architecture of eyes-open resting state EEG frequency activity (intrinsic frequency) from 64 channels in 105 SZ, 145 PBP probands and 56 healthy controls (HCs) from the multisite BSNIP (Bipolar-Schizophrenia Network on Intermediate Phenotypes) study. One million single-nucleotide polymorphisms (SNPs) were derived from DNA. We assessed eight data-driven EEG frequency activity derived from group-independent component analysis (ICA) in conjunction with a reduced subset of 10,422 SNPs through novel multivariate association using parallel ICA (para-ICA). Genes contributing to the association were examined collectively using pathway analysis tools. Para-ICA extracted five frequency and nine SNP components, of which theta and delta activities were significantly correlated with two different gene components, comprising genes participating extensively in brain development, neurogenesis and synaptogenesis. Delta and theta abnormality was present in both SZ and PBP, while theta differed between the two disorders. Theta abnormalities were also mediated by gene clusters involved in glutamic acid pathways, cadherin and synaptic contact-based cell adhesion processes. Our data suggest plausible multifactorial genetic networks, including novel and several previously identified (DISC1) candidate risk genes, mediating low frequency delta and theta abnormalities in psychoses. The gene clusters were enriched for biological properties affecting neural circuitry and involved in brain function and/or development.
Asunto(s)
Trastorno Bipolar/genética , Ritmo Delta/genética , Esquizofrenia/genética , Ritmo Teta/genética , Adulto , Trastorno Bipolar/fisiopatología , Encéfalo/crecimiento & desarrollo , Ondas Encefálicas/genética , Ondas Encefálicas/fisiología , Estudios de Casos y Controles , Adhesión Celular/genética , Ritmo Delta/fisiología , Electroencefalografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Neurogénesis/genética , Polimorfismo de Nucleótido Simple , Esquizofrenia/fisiopatología , Ritmo Teta/fisiología , Adulto JovenRESUMEN
We investigated the mechanism of formation and stability of self-assembled monolayers (SAMs) of methaneselenolate on Au(111) prepared by the immersion method in ethanolic solutions of dimethyl diselenide (DMDSe). The adsorbed species were characterized by electrochemical measurements and high-resolution photoelectron spectroscopy (HR-XPS). The importance of the headgroup on formation mechanism and the stability of the SAMs was addressed by comparatively studying methaneselenolate (MSe) and methanethiolate (MT) monolayers. Density Functional Theory (DFT) calculations were performed to identify the elementary reaction steps in the mechanisms of formation and decomposition of the monolayers. Reductive desorption and HR-XPS measurements indicated that a MSe monolayer is formed at short immersion times by the cleavage of the Se-Se bond of DMDSe. However, the monolayer decomposes at long immersion times at room temperature, as evidenced by the appearance of atomic Se on the surface. The decomposition is more pronounced for MSe than for MT monolayers. The MSe monolayer stability can be greatly improved by two modifications in the preparation method: immersion at low temperatures (-20 °C) and the addition of a reducing agent to the forming solution.
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Oro/química , Compuestos de Organoselenio/química , Compuestos de Sulfhidrilo/química , Adsorción , Técnicas Electroquímicas , Etanol/química , Espectroscopía de Fotoelectrones , Teoría Cuántica , Soluciones , Propiedades de Superficie , TemperaturaRESUMEN
High-resolution photoemission spectroscopy is used to characterize adlayers of ethane-, hexane-, and nonanedithiol molecules grown on Au(111) surfaces by the immersion method. The effect of using a reducing agent during and after the immersion to inhibit or eliminate S-S bonds is investigated. Our results demonstrate that immersion 24 h in millimolar dithiol ethanolic solutions gives rise to the formation of multilayers; this effect is more pronounced in the case of ethanedithiol, the shortest molecule. A post-treatment with a disulfide reducing agent is effective to produce monolayers of standing-up molecules; this effect is again more pronounced in the case of ethanedithiol. Finally, the immersion 24 h in a solution containing dithiol and the reducing agent gives an unexpected result: most molecules remain adsorbed in the lying-down configuration; in this case, the almost complete suppression of the standing-up phase occurs equally with the three types of molecules, which suggests that the formation of S-S bonds must be important for the lifting of the molecules.
Asunto(s)
Alcanos/química , Disulfuros/química , Oro/química , Sustancias Reductoras/química , Compuestos de Sulfhidrilo/química , Propiedades de SuperficieRESUMEN
Transient reactive oxygen species (ROS) production is currently proving to be an important mechanism in the regulation of intracellular signalling, but reports showing the involvement of ROS in important biological processes, such as cell differentiation, are scarce. In this study, we show for the first time that ROS production is required for megakaryocytic differentiation in K562 and HEL cell lines and also in human CD34(+) cells. ROS production is transiently activated during megakaryocytic differentiation, and such production is abolished by the addition of different antioxidants (such as N-acetyl cysteine, trolox, quercetin) or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. The inhibition of ROS formation hinders differentiation. RNA interference experiments have shown that a p22(phox)-dependent NADPH oxidase activity is responsible for ROS production. In addition, the activation of ERK, AKT and JAK2 is required for differentiation, but the activation of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase seems to be less important. When ROS production is prevented, the activation of these signalling pathways is partly inhibited. Taken together, these results show that NADPH oxidase ROS production is essential for complete activation of the main signalling pathways involved in megakaryocytopoiesis to occur. We suggest that this might also be important for in vivo megakaryocytopoiesis.
Asunto(s)
Megacariocitos/citología , NADPH Oxidasas/metabolismo , Antígenos CD34/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Cromanos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Janus Quinasa 2/metabolismo , Megacariocitos/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Compuestos Onio/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quercetina/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Atypical antipsychotics induce pre-diabetic symptoms in some but not all patients, characterized most notably by elevated weight. The side effect profiles of the various drugs in the class differ, however, raising the possibility of drug-specific mechanisms for similar side effects. We used physiogenomic analysis, an approach previously employed to study the genetics of drug and diet response, to discover and compare genetic associations with weight profiles observed in patients treated with olanzapine and risperidone as an approach to unraveling contrasting mechanistic features of both drugs. A total of 29 single nucleotide polymorphisms (SNPs) were selected from 13 candidate genes relevant to two potential pharmacological axes of psychotropic-related weight profiles, appetite peptides and peripheral lipid homeostasis. We applied physiogenomic analysis to a cross-section of 67 and 101 patients being treated with olanzapine and risperidone, respectively, and assessed genetic associations with the weight profiles. Weight profiles in patients treated with olanzapine were significantly associated with SNPs in the genes for apolipoprotein E, apolipoprotein A4 and scavenger receptor class B, member 1. Weight profiles in patients treated with risperidone were significantly associated with SNPs in the genes for leptin receptor, neuropeptide Y receptor Y5 and paraoxonase 1. These results are consistent with contrasting mechanisms for the weight profile of patients treated with these drugs. Genes associated with olanzapine weight profiles may be related to peripheral lipid homeostatic axes, whereas those associated with risperidone's may be related to brain appetite peptide regulation. Future physiogenomic studies will include neurotransmitter receptor SNPs and validation in independent samples.
Asunto(s)
Antipsicóticos/efectos adversos , Regulación del Apetito/genética , Metabolismo de los Lípidos/genética , Risperidona/efectos adversos , Aumento de Peso/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Antipsicóticos/uso terapéutico , Apolipoproteínas/efectos de los fármacos , Apolipoproteínas/genética , Regulación del Apetito/efectos de los fármacos , Arildialquilfosfatasa/efectos de los fármacos , Arildialquilfosfatasa/genética , Benzodiazepinas/efectos adversos , Benzodiazepinas/uso terapéutico , Estudios Transversales , Femenino , Genómica/métodos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Trastornos Mentales/tratamiento farmacológico , Persona de Mediana Edad , Olanzapina , Polimorfismo de Nucleótido Simple/genética , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/genética , Receptores de Leptina , Receptores de Neuropéptido Y/efectos de los fármacos , Receptores de Neuropéptido Y/genética , Risperidona/uso terapéutico , Receptores Depuradores de Clase B/efectos de los fármacos , Receptores Depuradores de Clase B/genética , Aumento de Peso/efectos de los fármacosRESUMEN
Cholesteryl ester transfer protein (CETP) plays a central role in high-density lipoprotein (HDL) metabolism. Single nucleotide polymorphisms (SNPs) and haplotypes in the CETP gene were determined in 98 patients with untreated dyslipidemias and analyzed for associations with plasma CETP and plasma lipids before and during statin treatment. Individual CETP SNPs and haplotypes were both significantly associated with CETP enzyme mass and activity. However, only certain CETP haplotypes, but not individual SNPs, significantly predicted the magnitude of change in HDL cholesterol (HDL-C) and triglycerides. After adjusting for covariates and multiple testing, the TTCAAA haplotype showed a gene-dose effect in predicting the HDL-C increase (P=0.03), while the TTCAAAGGG and AAAGGG haplotypes predicted a decrease in triglycerides (P=0.04 both). This is the first study to demonstrate that SNP haplotypes derived from allelic SNP combinations in the CETP gene were more informative than single SNPs in predicting the response to lipid-modifying therapy with statins.
Asunto(s)
Proteínas Portadoras/genética , Glicoproteínas , Haplotipos/genética , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Lípidos/genética , Anciano , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/genética , Proteínas de Transferencia de Ésteres de Colesterol , Estudios de Cohortes , Femenino , Variación Genética/genética , Humanos , Lípidos/sangre , Masculino , Persona de Mediana Edad , Valor Predictivo de las PruebasRESUMEN
Variation within genes has important implications for all biological traits. We identified 3899 single nucleotide polymorphisms (SNPs) that were present within 313 genes from 82 unrelated individuals of diverse ancestry, and we organized the SNPs into 4304 different haplotypes. Each gene had several variable SNPs and haplotypes that were present in all populations, as well as a number that were population-specific. Pairs of SNPs exhibited variability in the degree of linkage disequilibrium that was a function of their location within a gene, distance from each other, population distribution, and population frequency. Haplotypes generally had more information content (heterozygosity) than did individual SNPs. Our analysis of the pattern of variation strongly supports the recent expansion of the human population.
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Variación Genética , Haplotipos , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido Simple , Alelos , Animales , Pueblo Asiatico/genética , Población Negra/genética , Fosfatos de Dinucleósidos/genética , Evolución Molecular , Femenino , Heterocigoto , Hispánicos o Latinos/genética , Humanos , Masculino , Mutación , Pan troglodytes/genética , Población Blanca/genética , Cromosoma X/genéticaRESUMEN
The human beta(2)-adrenergic receptor gene has multiple single-nucleotide polymorphisms (SNPs), but the relevance of chromosomally phased SNPs (haplotypes) is not known. The phylogeny and the in vitro and in vivo consequences of variations in the 5' upstream and ORF were delineated in a multiethnic reference population and an asthmatic cohort. Thirteen SNPs were found organized into 12 haplotypes out of the theoretically possible 8,192 combinations. Deep divergence in the distribution of some haplotypes was noted in Caucasian, African-American, Asian, and Hispanic-Latino ethnic groups with >20-fold differences among the frequencies of the four major haplotypes. The relevance of the five most common beta(2)-adrenergic receptor haplotype pairs was determined in vivo by assessing the bronchodilator response to beta agonist in asthmatics. Mean responses by haplotype pair varied by >2-fold, and response was significantly related to the haplotype pair (P = 0.007) but not to individual SNPs. Expression vectors representing two of the haplotypes differing at eight of the SNP loci and associated with divergent in vivo responsiveness to agonist were used to transfect HEK293 cells. beta(2)-adrenergic receptor mRNA levels and receptor density in cells transfected with the haplotype associated with the greater physiologic response were approximately 50% greater than those transfected with the lower response haplotype. The results indicate that the unique interactions of multiple SNPs within a haplotype ultimately can affect biologic and therapeutic phenotype and that individual SNPs may have poor predictive power as pharmacogenetic loci.
Asunto(s)
Haplotipos , Regiones Promotoras Genéticas , Receptores Adrenérgicos beta 2/genética , Secuencia de Bases , Línea Celular Transformada , ADN/genética , Genotipo , Humanos , Filogenia , Polimorfismo de Nucleótido SimpleRESUMEN
Haplotype analysis has become increasingly important for the study of human disease as well as for reconstruction of human population histories. Computer programs have been developed to estimate haplotype frequencies statistically from marker phenotypes in unrelated individuals. However, there currently are few empirical reports on the accuracy of statistical estimates that must infer linkage phase. We have analyzed haplotypes at the CD4 locus on chromosome 12 that consist of a short tandem-repeat polymorphism and an Alu insertion/deletion polymorphism located 9.8 kb apart, in 398 individuals from 10 geographically diverse sub-Saharan African populations. Haplotype frequency estimates obtained using gene counting based on molecularly haplotyped (phase-known) data were compared with haplotype frequency estimates obtained using the expectation-maximization algorithm. We show that the estimated frequencies of common haplotypes do not differ significantly with the use of phase-known versus phase-unknown data. However, rare haplotypes are occasionally miscalled when their presence/absence must be inferred. Thus, for those research questions for which the common haplotypes are most important, frequency estimates based on the phase-unknown marker-typing results from unrelated individuals will be sufficient. However, in cases where knowledge of rare haplotypes is critical, molecular haplotyping will be necessary to determine linkage phase unambiguously.
Asunto(s)
Antígenos CD4/genética , Frecuencia de los Genes/genética , Haplotipos/genética , Estadística como Asunto/métodos , África del Sur del Sahara , Algoritmos , Elementos Alu/genética , Cromosomas Humanos Par 12/genética , Marcadores Genéticos/genética , Heterocigoto , Humanos , Desequilibrio de Ligamiento/genética , Mutación/genética , Polimorfismo Genético/genética , Proyectos de Investigación , Sensibilidad y Especificidad , Programas Informáticos , Secuencias Repetidas en Tándem/genéticaRESUMEN
A region-specific microdissection library originating from human chromosome 17q21, was constructed using the MboI linker-adaptor microcloning technique. DNA sequencing of 241 microclones resulted in the identification of 74 novel coding sequences, paralogs of known genes, and known, but previously unmapped, genes or expressed sequence tags that were "virtually" mapped to chromosome 17q21. By pooling the microclones as multiplexed hybridization probes, and by virtue of their origin on 17q21, we were able to identify approximately 150 P1 clones from the human Reference Library Data Base P1 Library that potentially map to chromosome 17q21. Verification of the 17q21 location of 16 P1 clones was accomplished by PCR analysis with STS primer pairs to known 17q21 genes or by FISH. Our results demonstrate the substantial advantage of combining the sequence analysis of microclones with multiplex hybridization strategies for gene discovery and mapping specific gene rich regions of the genome.
Asunto(s)
Cromosomas Humanos Par 17/genética , Biblioteca de Genes , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 17/ultraestructura , Clonación Molecular , Biología Computacional , Cartilla de ADN/genética , Bases de Datos Factuales , Humanos , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Haplotypes, combinations of polymorphic markers in a chromosome, are critical for genome diversity research. However, their utility in population samplings is compromised by uncertain linkage phase determinations from unrelated individuals. Molecular haplotyping accomplishes direct phase determination by generation of hemizygous templates from diploid genomic samples. We report molecular haplotyping by allele-specific long-range PCR of two markers 9.5 kb apart at the CD4 locus: a bi-allelic Alu deletion and a multi-allelic repeat. We verified CD4 molecular haplotypes by classical Mendelian analysis. Molecular haplotyping should prove useful in mapping disease genes and in establishing founder effects.
Asunto(s)
Alelos , Antígenos CD4/genética , Marcadores Genéticos , Haplotipos , Reacción en Cadena de la Polimerasa/métodos , Eliminación de Gen , Ligamiento Genético , Genotipo , Humanos , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
We have investigated the frequency distribution, across a broad range of geographically dispersed populations, of alleles of the polymorphic Alu insertion that occurs within the 8th intron of the tissue plasminogen, activator gene (PLAT). This Alu is a member of a recently derived subfamily of Alu elements that has been expanding during human evolution and continues to be transpositionally active. We used a "population tube" approach to screen 10 chromosomes from each of 19 human populations for presence or absence of this Alu in the PLAT locus and found that all tested populations are dimorphic for presence/absence of this insertion. We show that the previously published EcoRI, HincII, PstI, TaqI, and XmnI polymorphisms at the PLAT locus all result from insertion of this Alu and we use both restriction fragment length polymorphism and polymerase chain reaction analysis to examine the frequency of Alu(+) and Alu(-) alleles in a sample of 1003 individuals from 27 human populations and in 38 nonhuman primates. Nonhuman primates are monomorphic for the Alu(-) allele. Human populations differ substantially in allele frequency, and in several populations both alleles are common. Our results date the insertion event prior to the spread and diversification of modern humans.
Asunto(s)
Frecuencia de los Genes , Polimorfismo de Longitud del Fragmento de Restricción , Secuencias Repetitivas de Ácidos Nucleicos/genética , Activador de Tejido Plasminógeno/genética , Secuencia de Bases , Evolución Molecular , Pruebas Genéticas , Variación Genética/genética , Genética de Población , Humanos , Intrones/genética , Datos de Secuencia Molecular , Grupos Raciales/genéticaRESUMEN
A novel human chromosome locus-specific repetitive sequence was identified and characterized using arbitrary PCR. The repeat monomer consensus sequence is 100 bp long, and there are a minimum of 140 to 160 copies of the repetitive sequence per haploid human genome. The repetitive sequence is highly clustered on 20q12 within a 200- to 400-kb region. The highly polymorphic repeat array is inherited in a stable Mendelian fashion. Hybridization analysis revealed detectable conservation of the repeated element only among hominoids and Old World monkeys, where repeat arrangements are also polymorphic.
Asunto(s)
Genoma Humano , Hominidae/genética , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Secuencia de Bases , Southern Blotting , Cercopithecidae/genética , Mapeo Cromosómico , Secuencia de Consenso , ADN/genética , ADN/aislamiento & purificación , Femenino , Humanos , Células Híbridas , Masculino , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Técnica del ADN Polimorfo Amplificado Aleatorio , Homología de Secuencia de Ácido NucleicoRESUMEN
We have developed the population tube (poptube) system for sensitive detection and large-scale sampling of DNA sequence variation in several human populations of wide geographic distribution. In this methodology, genomic DNAs from five individuals in a population are PCR amplified en masse to maximize deliberately the chances of forming heteroduplexes among allelic variants. Interpopulation mixing is performed in a separate set of tubes containing one individual from each of five populations as well as a reference chimpanzee sample deliberately chosen to be different from all humans. Mismatches at sites of allelic variation retard the electrophoresis and reduce the stability of heteroduplex molecules. The products are electrophoresed on denaturing gradient gels where detection of heteroduplexes is accomplished readily. Using poptubes, we have discovered a rare variant in an otherwise highly conserved 440-bp segment in the long intron of the glucose-6-phosphate dehydrogenase (G6PD) gene. The polymorphism at this X-chromosome locus could be only detected in males by mixing samples, as homoduplexes for both alleles co-focus on denaturing gradient electrophoresis.