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1.
Prenat Diagn ; 39(11): 1011-1015, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31429096

RESUMEN

OBJECTIVE: To evaluate clinical performance of a new automated cell-free (cf)DNA assay in maternal plasma screening for trisomies 21, 18, and 13, and to determine fetal sex. METHOD: Maternal plasma samples from 1200 singleton pregnancies were analyzed with a new non-sequencing cfDNA method, which is based on imaging and counting specific chromosome targets. Reference outcomes were determined by either cytogenetic testing, of amniotic fluid or chorionic villi, or clinical examination of neonates. RESULTS: The samples examined included 158 fetal aneuploidies. Sensitivity was 100% (112/112) for trisomy 21, 89% (32/36) for trisomy 18, and 100% (10/10) for trisomy 13. The respective specificities were 100%, 99.5%, and 99.9%. There were five first pass failures (0.4%), all in unaffected pregnancies. Sex classification was performed on 979 of the samples and 99.6% (975/979) provided a concordant result. CONCLUSION: The new automated cfDNA assay has high sensitivity and specificity for trisomies 21, 18, and 13 and accurate classification of fetal sex, while maintaining a low failure rate. The study demonstrated that cfDNA testing can be simplified and automated to reduce cost and thereby enabling wider population-based screening.


Asunto(s)
Pruebas Prenatales no Invasivas/métodos , Trisomía/diagnóstico , Cromosomas Humanos Par 13 , Cromosomas Humanos Par 18 , Cromosomas Humanos Par 21 , Femenino , Humanos , Embarazo
2.
Nucleic Acids Res ; 39(2): e8, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21059679

RESUMEN

Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R(2) = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.


Asunto(s)
Análisis Mutacional de ADN/métodos , Genes Relacionados con las Neoplasias , Exones , Humanos
3.
Mol Cell Proteomics ; 9(2): 327-35, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19955079

RESUMEN

Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.


Asunto(s)
Proteínas Sanguíneas/análisis , Inmunoensayo/métodos , Microesferas , Ensayo de Inmunoadsorción Enzimática , Factor 15 de Diferenciación de Crecimiento/sangre , Humanos
4.
Nucleic Acids Res ; 38(16): e163, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20587504

RESUMEN

Alternative splicing creates diverse mRNA isoforms from single genes and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns along transcripts into a library of binary strings of nucleic acid tags that encode the exon composition of individual mRNA molecules. The exon inclusion pattern of each analyzed transcript is thus represented as binary data, and the abundance of different splice variants is registered by counts of individual molecules. The technique is illustrated in a model experiment by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits many genes to be analyzed in parallel and it will be valuable for elucidating the complex effects of combinatorial splicing.


Asunto(s)
Empalme Alternativo , Hibridación de Ácido Nucleico/métodos , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Sondas de ADN , Células HeLa , Humanos , Sitios de Empalme de ARN
5.
Nat Methods ; 5(2): 175-7, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18204456

RESUMEN

We describe a method, DNA array to protein array (DAPA), which allows the 'printing' of replicate protein arrays directly from a DNA array template using cell-free protein synthesis. At least 20 copies of a protein array can be obtained from a single DNA array. DAPA eliminates the need for separate protein expression, purification and spotting, and also overcomes the problem of long-term functional storage of surface-bound proteins.


Asunto(s)
Perfilación de la Expresión Génica/instrumentación , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Diseño de Equipo , Análisis de Falla de Equipo
6.
Nucleic Acids Res ; 36(8): e45, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18346972

RESUMEN

DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 10(5). Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas/análisis , ARN Mensajero/análisis , Actinas/genética , Actinas/metabolismo , Línea Celular , Sondas Moleculares , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Reacción en Cadena de la Polimerasa , Factor A de Crecimiento Endotelial Vascular/análisis
7.
Sci Rep ; 8(1): 4549, 2018 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-29540801

RESUMEN

Cell-free DNA analysis is becoming adopted for first line aneuploidy screening, however for most healthcare programs, cost and workflow complexity is limiting adoption of the test. We report a novel cost effective method, the Vanadis NIPT assay, designed for high precision digitally-enabled measurement of chromosomal aneuploidies in maternal plasma. Reducing NIPT assay complexity is achieved by using novel molecular probe technology that specifically label target chromosomes combined with a new readout format using a nanofilter to enrich single molecules for imaging and counting without DNA amplification, microarrays or sequencing. The primary objective of this study was to assess the Vanadis NIPT assay with respect to analytical precision and clinical feasibility. Analysis of reference DNA samples indicate that samples which are challenging to analyze with low fetal-fraction can be readily detected with a limit of detection determined at <2% fetal-fraction. In total of 286 clinical samples were analysed and 30 out of 30 pregnancies affected by trisomy 21 were classified correctly. This method has the potential to make cost effective NIPT more widely available with more women benefiting from superior detection and false positive rates.


Asunto(s)
Ácidos Nucleicos Libres de Células/sangre , Síndrome de Down/diagnóstico , Diagnóstico Prenatal/métodos , Imagen Individual de Molécula/métodos , Aneuploidia , Estudios de Casos y Controles , Análisis Costo-Beneficio , Femenino , Humanos , Embarazo , Diagnóstico Prenatal/economía , Estudios Prospectivos , Imagen Individual de Molécula/economía
8.
J Virol Methods ; 143(2): 200-6, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17451815

RESUMEN

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.


Asunto(s)
Enterovirus Humano B/aislamiento & purificación , Virus de la Fiebre Aftosa/aislamiento & purificación , Fiebre Aftosa/diagnóstico , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sondas de Oligonucleótidos , Infecciones por Rhabdoviridae/veterinaria , Enfermedades de los Porcinos/diagnóstico , Enfermedad Vesicular Porcina/diagnóstico , Virus de la Estomatitis Vesicular Indiana/aislamiento & purificación , Animales , Secuencia de Bases , Sondas de ADN , Enterovirus Humano B/genética , Virus de la Fiebre Aftosa/genética , Datos de Secuencia Molecular , Infecciones por Rhabdoviridae/diagnóstico , Porcinos , Virus de la Estomatitis Vesicular Indiana/genética
9.
PLoS One ; 7(7): e40405, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22808155

RESUMEN

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.


Asunto(s)
Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Animales , Extractos Celulares , Línea Celular , Humanos , Unión Proteica
10.
PLoS One ; 6(9): e25583, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21980495

RESUMEN

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.


Asunto(s)
Inmunoensayo/métodos , Proteómica/métodos , Análisis de Secuencia de ADN/métodos , Biomarcadores/sangre , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/genética , Humanos , Inmunoensayo/economía , Análisis Multivariante , Proteómica/economía , Análisis de Secuencia de ADN/economía , Factores de Tiempo
11.
Nat Methods ; 4(2): 135-7, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17179939

RESUMEN

The detection of weakly expressed proteins and protein complexes in biological samples represents a fundamental challenge. We have developed a new proximity-ligation strategy named 3PLA that uses three recognition events for the highly specific and sensitive detection of as little as a hundred molecules of the vascular endothelial growth factor (VEGF), the biomarkers troponin I, and prostate-specific antigen (PSA) alone or in complex with an inhibitor--demonstrating the versatility of 3PLA.


Asunto(s)
Inmunoensayo , Técnicas de Sonda Molecular , Proteínas/análisis , Anticuerpos/metabolismo , Biotina/metabolismo , Humanos , Hibridación de Ácido Nucleico , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa , Antígeno Prostático Específico/análisis , Sensibilidad y Especificidad , Estreptavidina/metabolismo , Troponina I/análisis , Células U937 , Factor A de Crecimiento Endotelial Vascular/análisis
12.
Electrophoresis ; 24(19-20): 3330-8, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-14595679

RESUMEN

We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.


Asunto(s)
Apirasa/metabolismo , Análisis Mutacional de ADN/métodos , Genes ras/genética , Melanoma/genética , Alelos , Análisis Mutacional de ADN/normas , Cartilla de ADN , Humanos , Melanoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa , Proto-Oncogenes Mas , Proto-Oncogenes/genética , Análisis de Secuencia de ADN/métodos
13.
Comp Funct Genomics ; 4(5): 525-30, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-18629012

RESUMEN

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.

14.
J Mol Recognit ; 17(3): 194-7, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15137029

RESUMEN

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.


Asunto(s)
Medicina Clínica/métodos , Genes/genética , Técnicas de Sonda Molecular/instrumentación , Proteínas/metabolismo , ARN Mensajero/metabolismo , Medicina Clínica/instrumentación , ADN/biosíntesis , ADN/genética , ADN/metabolismo , Humanos , ARN Mensajero/análisis , ARN Mensajero/genética
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