RESUMO
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.
Assuntos
Ouro/química , Compostos Organosselênicos/química , Compostos de Sulfidrila/química , Adsorção , Técnicas Eletroquímicas , Etanol/química , Espectroscopia Fotoeletrônica , Teoria Quântica , Soluções , Propriedades de Superfície , TemperaturaRESUMO
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.
Assuntos
Alcanos/química , Dissulfetos/química , Ouro/química , Substâncias Redutoras/química , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
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.
Assuntos
Dor Aguda , Bloqueio Nervoso , Humanos , Criança , Animais , Escorpiões , Bloqueio Nervoso/métodos , Serviço Hospitalar de Emergência , Ultrassonografia de Intervenção/métodosRESUMO
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.
RESUMO
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.
Assuntos
Variação Genética , Haplótipos , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Povo Asiático/genética , População Negra/genética , Fosfatos de Dinucleosídeos/genética , Evolução Molecular , Feminino , Heterozigoto , Hispânico ou Latino/genética , Humanos , Masculino , Mutação , Pan troglodytes/genética , População Branca/genética , Cromossomo X/genéticaRESUMO
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.
Assuntos
Materiais Biocompatíveis , DNA/administração & dosagem , Gelatina/química , Nanopartículas/química , Células 3T3 , Animais , Portadores de Fármacos , Células HeLa , Hemólise , Humanos , Camundongos , Protaminas/química , RatosRESUMO
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.
Assuntos
Transtorno Bipolar/genética , Ritmo Delta/genética , Esquizofrenia/genética , Ritmo Teta/genética , Adulto , Transtorno Bipolar/fisiopatologia , Encéfalo/crescimento & desenvolvimento , Ondas Encefálicas/genética , Ondas Encefálicas/fisiologia , Estudos de Casos e Controles , Adesão Celular/genética , Ritmo Delta/fisiologia , Eletroencefalografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Neurogênese/genética , Polimorfismo de Nucleotídeo Único , Esquizofrenia/fisiopatologia , Ritmo Teta/fisiologia , Adulto JovemRESUMO
Heat-soaked PCR (HS-PCR) is a method for enhancing amplification performed by heating the DNA sample at 94 degrees C in 90 microliters 1.1 x buffer for 30 min. A 10-microliters bolus of concentrated (10x) deoxynucleotides, Taq DNA polymerase and primers prepared without buffer is then added just prior to thermal cycling. We have investigated the application of this method in a variety of forensically important DNA samples and compared it with regular PCR (R-PCR). DNA samples extracted from bone, postmortem tissues, bloodstains and hair contained low concentrations of human DNA or were contaminated with either non- human DNA or hemoglobin degradation products. Optimal conditions for HS-PCR were determined for the 3' ApoB VNTR locus and applied to a centromeric repeat element and to a single-copy locus. HS-PCR consistently and reproducibly enhanced product yield and specificity over R-PCR at all three loci in the entire set of DNA samples. HS-PCR was also effective in overcoming the inhibitory effect of hemoglobin at concentrations that fully impeded R-PCR.
Assuntos
DNA/análise , Medicina Legal/métodos , Reação em Cadeia da Polimerase/métodos , DNA/sangue , Hemoglobinas , Temperatura Alta , Humanos , Mudanças Depois da MorteRESUMO
Nuclear DNA clones and sequence information derived from human genetic analyses were used to detect and characterize intra- and inter-species DNA variation at several nuclear loci in hominoids and cercopithecoids. Restriction fragment length polymorphisms were found at five loci among captive rhesus monkeys. Cross-species polymerase chain reaction (PCR) amplification detected an insertion within the beta-globin gene cluster in hylobatids. The combined use of cross-species PCR and denaturing gradient gel electrophoresis detected both species differences and intra-species polymorphism in the homeobox cluster 2 of hominoids. These results a) demonstrate that DNA clones and nucleotide sequence information from human molecular genetics can be used to facilitate studies of the molecular genetics of nonhuman primates, and b) document specific examples of intra- and inter-species molecular variability at several loci. © 1992 Wiley-Liss, Inc.
RESUMO
Human X and Y chromosome alpha-satellite sequences lying within higher order repeats were amplified by the polymerase chain reaction (PCR) in genomic deoxyribonucleic acid (DNA) isolated from blood, bone, and several other tissues and specimens of potential forensic science interest. X and Y sequences could be coamplified under some of the PCR conditions employed. Monomorphic sequences in the 3'-apolipoprotein B gene (designated "H") and in an alpha-satellite higher order repeat on Chromosome 17 (p17H8, D17Z1) were likewise amplified in the specimens. X and Y sequence amplification can provide information about the sex of origin. Amplification of the X, H, and D17Z1 sequences was found to be primate-specific among the common animals tested and can thus provide species of origin information about a specimen. The authors suggest that amplification of X and D17Z1 or H sequences might provide "relaxed" and "stringent" controls for appropriate PCR amplification tests on forensic science specimens. Testing was carried out using PCR protocols that employed Thermophilus aquaticus (Taq) and Thermus flavis (Replinase) thermostable DNA polymerases.
Assuntos
DNA/química , Primatas/genética , Análise para Determinação do Sexo , Cromossomo X/química , Cromossomo Y/química , Animais , Sequência de Bases , Feminino , Amplificação de Genes , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Especificidade da EspécieRESUMO
Deoxyribonucleic acid (DNA) was isolated from a number of spongy and compact human bone tissue specimens, and the yield was estimated on a "per milligram of starting tissue" basis. DNA was, in addition, isolated from a number of corresponding blood and bone tissue specimens. Spectrophotofluorometry and ethidium bromide visualization on minigels were used to estimate the quantity and degree of degradation of DNA. The DNA from several blood-bone pairs is shown to give concordant restriction fragment length polymorphism (RFLP) typing results by two different typing protocols with five different single-locus probes. DNA from several additional blood-bone pairs is shown to give concordant results for human leucocyte antigen (HLA)-DQ alpha phenotypes following polymerase chain reaction (PCR) amplification and hybridization to specific allele-specific oligonucleotide (ASO) probes, and for the variable numbers of tandem repeats (VNTR) length polymorphisms 3' to the human apolipoprotein B (APOB) gene following PCR amplification with specific primers and analysis of the products by electrophoresis and ethidium bromide visualization.
Assuntos
Osso e Ossos/química , DNA/análise , Marcadores Genéticos , Polimorfismo de Fragmento de Restrição , Sequência de Bases , Manchas de Sangue , DNA/sangue , DNA/química , Eletroforese em Gel de Ágar , Etídio , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Espectrometria de FluorescênciaRESUMO
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)
Assuntos
Humanos , Masculino , Criança , Nervo Isquiático , Picadas de Escorpião , Analgésicos , Emergências , Dor Aguda , Anestesia por Condução , Pediatria , Pacientes Internados , Exame Físico , Anestesiologia , UltrassonografiaRESUMO
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.
Assuntos
Megacariócitos/citologia , NADPH Oxidases/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Cromanos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Janus Quinase 2/metabolismo , Megacariócitos/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Oniocompostos/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quercetina/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Antipsicóticos/efeitos adversos , Regulação do Apetite/genética , Metabolismo dos Lipídeos/genética , Risperidona/efeitos adversos , Aumento de Peso/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Antipsicóticos/uso terapêutico , Apolipoproteínas/efeitos dos fármacos , Apolipoproteínas/genética , Regulação do Apetite/efeitos dos fármacos , Arildialquilfosfatase/efeitos dos fármacos , Arildialquilfosfatase/genética , Benzodiazepinas/efeitos adversos , Benzodiazepinas/uso terapêutico , Estudos Transversais , Feminino , Genômica/métodos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Transtornos Mentais/tratamento farmacológico , Pessoa de Meia-Idade , Olanzapina , Polimorfismo de Nucleotídeo Único/genética , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Receptores para Leptina , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Receptores de Neuropeptídeo Y/genética , Risperidona/uso terapêutico , Receptores Depuradores Classe B/efeitos dos fármacos , Receptores Depuradores Classe B/genética , Aumento de Peso/efeitos dos fármacosRESUMO
Addition of dideoxyribonucleotides during the exponential phase of the PCR should result in the synthesis of two complementary sequence ladders. We have explored this hypothesis to develop coupled amplification and sequencing of genomic DNA. Coupled amplification and sequencing is a biphasic method for sequencing both strands of template as they are amplified. Stage I selects and amplifies a single target from the genomic DNA sample. Stage II accomplishes the sequencing as well as additional amplification of the target using aliquots from the stage I reaction mixed with end-labeled primer and dideoxynucleotides. We have successfully applied coupled amplification and sequencing to a 300-base-pair fragment 4 kilobases upstream from HOX2B directly from human whole genomic DNA.
Assuntos
Sequência de Bases , DNA/genética , Genes Homeobox , Genes , Reação em Cadeia da Polimerase/métodos , Cromossomos Humanos Par 17 , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Sondas de OligonucleotídeosRESUMO
Coupled amplification and sequencing (CAS) allows a segment of DNA to be sequenced directly from genomic DNA. An initial PCR amplification stage selects and amplifies the target. During a subsequent stage both strands of the target segment are sequenced simultaneously and amplified further. We show that CAS can readily identify variant base pairs. Genotyping of a population for known sequence variation can be achieved simply and directly from genomic DNA of each organism by performing CAS only for the variant bases. The procedure supercedes development and optimization of alternative typing assays based on oligonucleotide hybridization or ligation. In addition, we show that competitive oligonucleotide priming with allelic primers can be readily performed in concert with the second stage of CAS. The combination of techniques allows sequencing of a single chromosome from a heterozygous genomic sample and direct haplotyping of the polymorphism at the priming site with any others encompassed within the amplified segment.
Assuntos
Genes Homeobox/genética , Testes Genéticos/métodos , Globinas/genética , Família Multigênica/genética , Polimorfismo Genético/genética , Animais , Sequência de Bases , DNA/genética , Eletroforese , Feminino , Genoma , Genótipo , Haplótipos/genética , Humanos , Masculino , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Pan troglodytes/genética , Reação em Cadeia da Polimerase , Especificidade da EspécieRESUMO
We have investigated the capability of PCR to amplify a specific locus from each template in a mixture of allelic DNAs. Modeling experiments employed a 300-bp HOX2B segment as target and utilized, as distinct template "alleles," genomic DNAs from 1 human and from 2 chimpanzees known to differ in sequence at the target. Two modes were used: (1) mixtures of PCR products that had been previously amplified individually from a single template and (2) PCR amplification en masse from composite human/chimpanzee genomic DNA templates. Products generated by either mode were separated by denaturing gradient gel electrophoresis (DGGE). Detection of a "trace" allele mixed with a "dominant" one was possible by simple ethidium bromide staining of the gel up to a sensitivity of 1 part in 20. A balanced mixture was represented by 5:5 and 4:3:3 mixtures of allelic PCR products or genomic templates; an uneven mixture of dominant and trace alleles, by 9:1 and 8:1:1 mixtures. For 5:5, two homoduplex bands and two heteroduplex bands of equal intensity were generated. For 9:1, the trace homoduplex disappears whereas the two heteroduplexes are easily visible. For 8:1:1, four heteroduplexes and one homoduplex were observed; homoduplexes and heteroduplexes formed from the trace alleles were not visible. These experiments demonstrate that PCR can amplify mixed allelic templates in direct proportion to the stoichiometric fraction of each template. Trace species are captured as heteroduplexes with the most abundant species and are clearly displaced on denaturing gradient gels from the dominant homoduplex species. Our analytical studies can be applied to analysis of sequence variants in DNA collected from cancerous or infected tissues.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
DNA/metabolismo , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Moldes Genéticos , Animais , Eletroforese em Gel de Poliacrilamida , Genes Homeobox , Humanos , Desnaturação de Ácido Nucleico , Pan troglodytes/genética , Especificidade da EspécieRESUMO
A method has been developed for simultaneous radiolabeling and amplification of DNA hybridization probes. The method is termed cycled primer extension (CPE). CPE is a series of temperature-driven reactions in which template DNA is successively denatured and extended by a thermostable primer-dependent DNA polymerase. The primers consist of semirandom nanomers of the form 5'-NNN NNN (G/C)(G/C)(G/C)-3'. These nanomers have the capacity to anneal to any template DNA and serve as initial anchors for extension at the high temperatures required for Taq DNA polymerase activity. CPE cycles consist of 94 degrees C denaturation, annealing of primers to template upon ramping to 24 degrees C, and gradual extension of the primer along the template as temperature is ramped back to 94 degrees C. Labeling efficiency with [32P]dCTP was examined and optimized as determined by the relation to ratios of radiolabeled to unlabeled dCTP, by number of cycles, and by primer composition and sequence. CPE probes can be generated without regard to size or sequence of template and have a high specific activity (approximately 10(9) dpm/micrograms). With CPE, hybridization signals equivalent to those from random primed probes are routinely obtained with initial template amounts as low as 1 ng.
Assuntos
Sondas de DNA , Técnicas de Sonda Molecular , DNA/biossíntese , Estudos de Avaliação como Assunto , Amplificação de Genes , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Radioisótopos de Fósforo , TemperaturaRESUMO
In a recent paper we have shown that DNA haplotypes of multiply heterozygous individuals can be resolved directly by polymerase-chain-reaction (PCR) amplification of a single molecule of genomic template. Our method (the single-molecule-dilution [SMD] method) relies on the stochastic separation of maternal and paternal alleles at high dilution. The stochasticity of separation and the potential for DNA shearing (which could separate the loci of interest) are two factors that can compromise the results of the experiment. This paper explores the consequences of these two factors and shows that the SMD method can be expected to work very reliably even in the presence of a moderate amount of DNA shearing.
Assuntos
Haplótipos , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Alelos , DNA , Heterozigoto , Humanos , ProbabilidadeRESUMO
We have developed a reliable method for the direct resolution of haplotypes or linkage phase from individuals who are multiply heterozygous in a given genomic region. The method is based on single-molecule dilution (SMD) of genomic template and amplification via biphasic polymerase chain reaction (booster PCR). We have verified the feasibility of the SMD method for a highly polymorphic region within the beta-globin cluster by analysis of triply heterozygous individuals of known haplotype. This approach should be useful in many studies in population or evolutionary genetics and in a variety of clinical settings.