Simplified buccal DNA extraction with FTA® Elute Cards

Wolfgramm, Eldamária de Vargas; Carvalho, Fernanda Magri de; Aguiar, Vitor R. C.; Sartori, Mariana Penha De Nadai; Hirschfeld-Campolongo, Gabriela C.R.; Tsutsumida, Weslley M.; Louro, Iúri Drumond

doi:10.1016/j.fsigen.2008.11.008

Cited by 34 publications

(16 citation statements)

References 12 publications

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“…As an alternative to the usual venipuncture technique, several methods have been described that use DBS from finger-puncture or oral swabs collected on DNA-cards [22], [23], [24], [25]. Buccal mucosa cell sampling is almost completely non-invasive and painless, reducing compliance issues.…”

Section: Discussionmentioning

confidence: 99%

Detection of IL28B SNP DNA from Buccal Epithelial Cells, Small Amounts of Serum, and Dried Blood Spots

et al. 2012

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Background & AimsPoint mutations in the coding region of the interleukin 28 gene (rs12979860) have recently been identified for predicting the outcome of treatment of hepatitis C virus infection. This polymorphism detection was based on whole blood DNA extraction. Alternatively, DNA for genetic diagnosis has been derived from buccal epithelial cells (BEC), dried blood spots (DBS), and genomic DNA from serum. The aim of the study was to investigate the reliability and accuracy of alternative routes of testing for single nucleotide polymorphism allele rs12979860CC.MethodsBlood, plasma, and sera samples from 200 patients were extracted (400 µL). Buccal smears were tested using an FTA card. To simulate postal delay, we tested the influence of storage at ambient temperature on the different sources of DNA at five time points (baseline, 48 h, 6 days, 9 days, and 12 days)ResultsThere was 100% concordance between blood, plasma, sera, and BEC, validating the use of DNA extracted from BEC collected on cytology brushes for genetic testing. Genetic variations in HPTR1 gene were detected using smear technique in blood smear (3620 copies) as well as in buccal smears (5870 copies). These results are similar to those for whole blood diluted at 1/10. A minimum of 0.04 µL, 4 µL, and 40 µL was necessary to obtain exploitable results respectively for whole blood, sera, and plasma. No significant variation between each time point was observed for the different sources of DNA. IL28B SNPs analysis at these different time points showed the same results using the four sources of DNA.ConclusionWe demonstrated that genomic DNA extraction from buccal cells, small amounts of serum, and dried blood spots is an alternative to DNA extracted from peripheral blood cells and is helpful in retrospective and prospective studies for multiple genetic markers, specifically in hard-to-reach individuals.

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Section: Discussionmentioning

confidence: 99%

Detection of IL28B SNP DNA from Buccal Epithelial Cells, Small Amounts of Serum, and Dried Blood Spots

et al. 2012

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“…Se observa que las tarjetas FTA TM Genecard Whatman TM (figura 3) presentan mejores perfiles genéticos, puesto que muestran mayor altura de los picos; esto posiblemente es debido a que estas tarjetas están diseñadas e impregnadas con fórmula Kruskal-Wallis chi-cuadrado = 217,7487 df = 4, p-valor < 0,001 química patentada que lisa las membranas celulares y desnaturaliza las enzimas que degradan el ADN, y el ADN queda atrapado en la matrix de celulosa protegido de las condiciones ambientales (de Vargas Wolfgramm et al 2009). A diferencia de las tarjetas FTA, las tarjetas papel de cromatografía (Chromatography paper Whatman TM ) y papel filtro (Rundfilter/Filter paper circles 125 mm) contienen soluciones químicas para lisar las células y conservar el ADN; es probable que por esta razón la altura del pico es menor.…”

Section: Amplificación De Adn Según El Diámetro Del Círculounclassified

“…La extracción del ADN, es el paso inicial en los procedimientos de un laboratorio de genética, consiste en aislar la molécula de ADN de cualquier tipo de célula nucleada, en condiciones de alta calidad, pureza y buena cantidad, que permita su caracterización genética (ChaconCortes et al 2012); se utilizan diferentes metodologías como el Chelex ® 100 y QIAGEN DNA Investigator Kit (Phillips et al 2012, Ps et al 1991) o el reactivo de purificación FTA (de Vargas Wolfgramm et al 2009, entre otros).…”

Section: Introductionunclassified

Variación de perfiles genéticos obtenidos por PCR con y sin extracción de ADN a partir de manchas de sangre

et al. 2017

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ResumenLa PCR sin extracción es una herramienta clave en investigaciones forenses, tratándose de una de las opciones a elegir cuando se tienen muestras con baja cantidad o calidad del ADN. El objetivo de este estudio fue determinar si hay diferencias entre los perfiles genéticos obtenidos por PCR i) en muestras de ADN extraídas con Chelex ® 100, ii) en muestras de ADN obtenidas con el reactivo de purificación de muestras en tarjetas FTA, y iii) por la PCR muestras sin extracción, empleando el kit comercial AmpFLSTR ® Identifiler ® , utilizado de rutina en el laboratorio IdentiGEN de la Universidad de Antioquia. Manchas de sangre almacenadas en tarjetas FTA TM Genecard Whatman TM , papel de Cromatografía y papel filtro, se amplificaron por PCR sin extracción utilizando los buffers Ezway TM , PCRboost TM , STRboost TM y Buffer 5X Colorless Go Taq ® Flexi. Los perfiles obtenidos se analizaron por medio de electroforesis capilar. Los resultados muestran que con los buffers utilizados se obtiene la amplificación de todos los marcadores STRs de las muestras almacenadas en los tres diferentes soportes. Esto permite concluir, que utilizando la PCR sin extracción, además de reducirse los costos y el tiempo de procesamiento de las muestras, se logra obtener perfiles genéticos que cumplan los criterios de calidad establecidos por el laboratorio, generando resultados de manera más eficiente al no ser necesario realizar la extracción y purificación del ADN.Palabras claves: PCR sin extracción, extracción de ADN, genética forense. AbstractThe PCR without extraction is a key tool in forensic investigation, being a good option when samples with small amounts or degraded DNA are available. The objective of this study was to compare genetic profiles obtained by PCR using: i) DNA samples extracted using Chelex ® 100, ii) DNA samples extracted using an FTA purification reagent, and iii) samples without extraction using the commercial kits AmpFLSTR ® Identifiler ® , routinely employed in the laboratory IdentiGEN of the University of Antioquia. Blood stains were collected in FTA cards Genecard Whatman TM , chromatography paper, and in filter papers. PCR without extraction was performed using the buffers Ezway TM , PCRboost TM , STRboost TM , and Buffer 5X Colorless Go Taq ® Flexi. The profiles were obtained by capillary electrophoresis. The results show that, using the above-mentioned buffers, all STR markers were amplified, for samples collected in the three different types of support. In conclusion, the PCR without extraction, apart from reducing the cost and time needed for processing the samples, it allows obtaining genetic profiles that meet the quality criteria established by the laboratory, in a more efficiently way, since DNA extraction and purification steps are eliminated.

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“…This need has motivated the community (government, academia and industry) to work collaboratively to develop and validate standard DNA typing kits that contain the reagents necessary to type core identity genetic markers and the concomitant automation of a number of processes (extraction, quantitation, amplification and to some degree interpretation of the results) to facilitate typing. The ability, for example, to type a reference blood or buccal sample without extracting the DNA away from interfering compounds would not have been imagined possible a few years ago and yet today such technology is readily available [2,3]. …”

Section: Introduction: Forensic Genetics and Challenging Samplesmentioning

confidence: 99%

Genetic Identification of Missing Persons: DNA Analysis of Human Remains and Compromised Samples

et al. 2012

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Human identification has made great strides over the past 2 decades due to the advent of DNA typing. Forensic DNA typing provides genetic data from a variety of materials and individuals, and is applied to many important issues that confront society. Part of the success of DNA typing is the generation of DNA databases to help identify missing persons and to develop investigative leads to assist law enforcement. DNA databases house DNA profiles from convicted felons (and in some jurisdictions arrestees), forensic evidence, human remains, and direct and family reference samples of missing persons. These databases are essential tools, which are becoming quite large (for example the US Database contains 10 million profiles). The scientific, governmental and private communities continue to work together to standardize genetic markers for more effective worldwide data sharing, to develop and validate robust DNA typing kits that contain the reagents necessary to type core identity genetic markers, to develop technologies that facilitate a number of analytical processes and to develop policies to make human identity testing more effective. Indeed, DNA typing is integral to resolving a number of serious criminal and civil concerns, such as solving missing person cases and identifying victims of mass disasters and children who may have been victims of human trafficking, and provides information for historical studies. As more refined capabilities are still required, novel approaches are being sought, such as genetic testing by next-generation sequencing, mass spectrometry, chip arrays and pyrosequencing. Single nucleotide polymorphisms offer the potential to analyze severely compromised biological samples, to determine the facial phenotype of decomposed human remains and to predict the bioancestry of individuals, a new focus in analyzing this type of markers.

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Simplified buccal DNA extraction with FTA® Elute Cards

Cited by 34 publications

References 12 publications

Detection of IL28B SNP DNA from Buccal Epithelial Cells, Small Amounts of Serum, and Dried Blood Spots

Detection of IL28B SNP DNA from Buccal Epithelial Cells, Small Amounts of Serum, and Dried Blood Spots

Variación de perfiles genéticos obtenidos por PCR con y sin extracción de ADN a partir de manchas de sangre

Genetic Identification of Missing Persons: DNA Analysis of Human Remains and Compromised Samples

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