RESUMO
PURPOSE: The aim of the study was to evaluate the diagnostic accuracy of an informatics-based, noninvasive, prenatal paternity test using array-based single-nucleotide polymorphism measurements of cell-free DNA isolated from maternal plasma. METHODS: Blood samples were taken from 21 adult pregnant women (with gestational ages between 6 and 21 weeks), and a genetic sample was taken from the corresponding biological fathers. Paternity was confirmed by genetic testing of the infant, products of conception, control of fertilization, and/or preimplantation genetic diagnosis during in vitro fertilization. Parental DNA samples and maternal plasma cell-free DNA were amplified and analyzed using a HumanCytoSNP-12 array. An informatics-based method measured single-nucleotide polymorphism data, confirming or rejecting paternity. Each plasma sample with a sufficient fetal cell-free DNA fraction was independently tested against the confirmed father and 1,820 random, unrelated males. RESULTS: One of the 21 samples had insufficient fetal cell-free DNA. The test correctly confirmed paternity for the remaining 20 samples (100%) when tested against the biological father, with P values of <10(-4). For the 36,400 tests using an unrelated male as the alleged father, 99.95% (36,382) correctly excluded paternity and 0.05% (18) were indeterminate. There were no miscalls. CONCLUSION: A noninvasive paternity test using informatics-based analysis of single-nucleotide polymorphism array measurements accurately determined paternity early in pregnancy.
Assuntos
Biologia Computacional , Paternidade , Diagnóstico Pré-Natal , Adulto , Sistema Livre de Células , Biologia Computacional/métodos , DNA/sangue , Feminino , Testes Genéticos , Idade Gestacional , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Gravidez , Diagnóstico Pré-Natal/métodosRESUMO
OBJECTIVE: This study aims to develop a noninvasive prenatal test on the basis of the analysis of cell-free DNA in maternal blood to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y. METHODS: A total of 166 samples from pregnant women, including 11 trisomy 21, three trisomy 18, two trisomy 13, two 45,X, and two 47,XXY samples, were analyzed using an informatics-based method. Cell-free DNA from maternal blood was isolated, amplified using a multiplex polymerase chain reaction (PCR) assay targeting 11,000 single nucleotide polymorphisms on chromosomes 13, 18, 21, X, and Y in a single reaction, and sequenced. A Bayesian-based maximum likelihood statistical method was applied to determine the chromosomal count of the five chromosomes interrogated in each sample, along with a sample-specific calculated accuracy for each test result. RESULTS: The algorithm correctly reported the chromosome copy number at all five chromosomes in 145 samples that passed a DNA quality test, for a total of 725/725 correct calls. The average calculated accuracy for these samples was 99.92%. Twenty-one samples did not pass the DNA quality test. CONCLUSIONS: This informatics-based method noninvasively detected fetuses with trisomy 13, 18, and 21, 45,X, and 47,XXY with high sample-specific calculated accuracies for each individual chromosome and across all five chromosomes.
Assuntos
Cromossomos Humanos , Testes para Triagem do Soro Materno , Transtornos dos Cromossomos Sexuais/diagnóstico , Trissomia/diagnóstico , Feminino , Humanos , Cariótipo , Masculino , Gravidez , Aberrações dos Cromossomos SexuaisRESUMO
Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.
Assuntos
Genes Neoplásicos , Reação em Cadeia da Polimerase/métodos , Primers do DNA , Éxons , Genômica/métodos , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the avian influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for detection. The outstanding feature of this assay is its capability to designate both the HA and the NA of an AIV sample from a single reaction mixture. A panel of 77 influenza virus strains was tested representing the entire assortment of the two antigens. One hundred percent (77/77) of the samples tested were identified as AIV, and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Influenza Aviária/virologia , Neuraminidase/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Orthomyxoviridae/classificação , Orthomyxoviridae/genética , Animais , Primers do DNA/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Aves Domésticas , Sensibilidade e Especificidade , Proteínas Virais/genéticaRESUMO
The World Organization for Animal Health (Office International des Epizooties, OIE) includes the diseases caused by foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV), as "Diseases Notifiable to the OIE". Foot-and-mouth disease (FMD) outbreaks have severe economical as well as social effects and cannot be differentiated from the diseases caused by the other two viruses on the basis of clinical symptoms. Efficient laboratory techniques are therefore required for detection and identification of the viruses causing similar vesicular symptoms in swine. A rapid method is described using padlock probes and microarrays to detect simultaneously and differentiate the three viruses in a single reaction, as well as providing serotype information in cases of VSV infection. The padlock probe/microarray assay detected successfully and identified 39 cDNA samples of different origin representing the three viruses. The results were in complete agreement with identities and serotypes determined previously. This novel virus detection method is discussed in terms of usefulness and further development.
Assuntos
Enterovirus Humano B/isolamento & purificação , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/diagnóstico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de Oligonucleotídeos , Infecções por Rhabdoviridae/veterinária , Doenças dos Suínos/diagnóstico , Doença Vesicular Suína/diagnóstico , Vírus da Estomatite Vesicular Indiana/isolamento & purificação , Animais , Sequência de Bases , Sondas de DNA , Enterovirus Humano B/genética , Vírus da Febre Aftosa/genética , Dados de Sequência Molecular , Infecções por Rhabdoviridae/diagnóstico , Suínos , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.
Assuntos
Perfilação da Expressão Gênica/métodos , Técnicas de Sonda Molecular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Células Cultivadas , Cromossomos Humanos Par 6/genética , Análise Mutacional de DNA/métodos , Etiquetas de Sequências Expressas , Genótipo , Humanos , Controle de Qualidade , Análise de Sequência de DNA/métodosRESUMO
Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.
Assuntos
Técnicas de Diagnóstico Molecular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de Oligonucleotídeos/química , Doenças das Plantas/microbiologia , Animais , Fungos/genética , Fungos/isolamento & purificação , Nematoides/genética , Nematoides/isolamento & purificação , Oomicetos/genética , Oomicetos/isolamento & purificação , Doenças das Plantas/parasitologiaRESUMO
Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de Oligonucleotídeos/genética , Polimorfismo de Nucleotídeo Único/genética , Adenosina Trifosfatases/genética , Alelos , Proteínas de Transporte de Cátions/genética , ATPases Transportadoras de Cobre , DNA/química , DNA/genética , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , Genótipo , Humanos , Mutação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodosRESUMO
Padlock probes are molecular tools that combine highly specific target sequence recognition with the potential for multiplexed analysis of large sets of target DNA or RNA sequences. In this brief review, we exemplify the ability of these probes to distinguish single-nucleotide target sequence variants. We further discuss means to detect the location of target sequences in situ, and to amplify reacted padlock probes via rolling-circle replication, as well as to sort reaction products on tag-arrays. We argue that the probes have the potential to render high-throughput genetic analyses precise and affordable.
Assuntos
Sondas de DNA/química , Sondas de DNA/genética , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , DNA/biossíntese , DNA/química , DNA/genética , DNA Ligases/metabolismo , Sondas de DNA/metabolismo , Replicação do DNA , Modelos Moleculares , RNA/química , RNA/genética , Sensibilidade e Especificidade , Especificidade por SubstratoRESUMO
OBJECTIVE: To characterize chromosomal error types and parental origin of aneuploidy in cleavage-stage embryos using an informatics-based technique that enables the elucidation of aneuploidy-causing mechanisms. DESIGN: Analysis of blastomeres biopsied from cleavage-stage embryos for preimplantation genetic screening during IVF. SETTING: Laboratory. PATIENT(S): Couples undergoing IVF treatment. INTERVENTION(S): Two hundred seventy-four blastomeres were subjected to array-based genotyping and informatics-based techniques to characterize chromosomal error types and parental origin of aneuploidy across all 24 chromosomes. MAIN OUTCOME MEASURE(S): Chromosomal error types (monosomy vs. trisomy; mitotic vs. meiotic) and parental origin (maternal vs. paternal). RESULT(S): The rate of maternal meiotic trisomy rose significantly with age, whereas other types of trisomy showed no correlation with age. Trisomies were mostly maternal in origin, whereas paternal and maternal monosomies were roughly equal in frequency. No examples of paternal meiotic trisomy were observed. Segmental error rates were found to be independent of maternal age. CONCLUSION(S): All types of aneuploidy that rose with increasing maternal age can be attributed to disjunction errors during meiosis of the oocyte. Chromosome gains were predominantly maternal in origin and occurred during meiosis, whereas chromosome losses were not biased in terms of parental origin of the chromosome. The ability to determine the parental origin for each chromosome, as well as being able to detect whether multiple homologs from a single parent were present, allowed greater insights into the origin of aneuploidy.
Assuntos
Aneuploidia , Blastômeros/patologia , Aberrações Cromossômicas , Fertilização in vitro , Diagnóstico Pré-Implantação , Adulto , Técnicas de Cultura Embrionária , Pai , Feminino , Predisposição Genética para Doença , Humanos , Cariotipagem , Masculino , Idade Materna , Meiose/genética , Pessoa de Meia-Idade , Mitose/genética , Mosaicismo , Mães , Medição de Risco , Fatores de RiscoRESUMO
BACKGROUND: Detection of expanded T-cell clones, identified by their receptor (TCR) repertoires, can assist diagnosis and guide therapy in infectious, inflammatory, and autoimmune conditions as well as in tumor immunotherapy. Analysis of tumor-infiltrating lymphocytes often reveals preferential use of one or a few TCR V beta genes, compared with peripheral blood, indicative of a clonal response against tumor antigens. METHODS: To simultaneously measure the relative expression of all V beta gene families, we combined highly specific and sensitive oligonucleotide reagents, called padlock probes, with a microarray read-out format. T-Cell cDNA was combined with a pool of V beta subfamily-specific padlock probes. Reacted probes were selectively amplified and the products hybridized to a microarray, from which the V beta subfamily distribution in each sample could be determined relative to a control sample. RESULTS: In lymphocytes stimulated with the superantigen staphylococcal enterotoxin B, we detected expansions at the mRNA level of TCR subfamilies previously shown to respond to staphylococcal enterotoxin B. Expansions of the same V beta families could also be detected by flow cytometry. In samples from two bladder cancer patients, we detected predominant representations of specific V beta subfamilies in both tumor-infiltrating lymphocytes and in the draining lymph nodes, but not in non-tumor-draining lymph nodes or peripheral blood. Several expression profiles from draining lymph nodes in patients with malignant melanoma were divergent from profiles seen in non-tumor-draining lymph nodes. CONCLUSION: Padlock probe-based parallel analysis of TCR V beta gene distributions provides an efficient method for screening multiple samples for T-cell clonal expansions with reduced labor and time of analysis compared with traditional methods.
Assuntos
Perfilação da Expressão Gênica , Genes Codificadores dos Receptores de Linfócitos T , Ativação Linfocitária , Melanoma/diagnóstico , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Neoplasias da Bexiga Urinária/diagnóstico , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , DNA Complementar/análise , Enterotoxinas/imunologia , Humanos , Linfócitos/metabolismo , Melanoma/genética , Melanoma/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Padrões de Referência , Staphylococcus aureus/imunologia , Superantígenos/imunologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/imunologiaRESUMO
We present a tightly controlled process for strand-specific amplification of circularized DNA molecules. Tandem repeated complements of DNA circles are generated by rolling-circle replication, and converted to monomer circles of opposite polarity to that of the starting material. These circles are then subjected to one more round of rolling-circle replication and circularization, and the process can be further repeated. The method can be directed to produce single-stranded circular or linear monomers, or linear concatemers of the desired polarity. The reaction is not product inhibited, and can yield approximately 100-fold higher concentrations of monomer products than PCR. Each generation of the amplification process proceeds in a linear fashion, ensuring precise quantification. The procedure is suitable for parallel amplification of large numbers of DNA circles, because the few cycles and the robust reaction mechanism preserves the proportion of amplified molecules. We demonstrate the utility of the method for multiplexed genotyping of polymorphic loci and for quantitative DNA analysis.
Assuntos
DNA Circular/genética , DNA/genética , Sequência de Bases , DNA/química , Replicação do DNA/genética , DNA Circular/química , Amplificação de Genes , Doenças Genéticas Inatas/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.
Assuntos
Medicina Clínica/métodos , Genes/genética , Técnicas de Sonda Molecular/instrumentação , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Medicina Clínica/instrumentação , DNA/biossíntese , DNA/genética , DNA/metabolismo , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
Highly specific high-throughput assays will be required to take full advantage of the accumulating information about the macromolecular composition of cells and tissues, in order to characterize biological systems in health and disease. We discuss the general problem of detection specificity and present the approach our group has taken, involving the reformatting of analogue biological information to digital reporter segments of genetic information via a series of DNA ligation assays. The assays enable extensive, coordinated analyses of the numbers and locations of genes, transcripts and protein.