High efficiency DNA extraction from bone by total demineralization

Loreille, Odile; Diegoli, Toni M.; Irwin, Jodi A.; Coble, Michael D.; Parsons, Thomas J.

doi:10.1016/j.fsigen.2007.02.006

Cited by 252 publications

(148 citation statements)

References 4 publications

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“…Los resultados del análisis de varianza indicaron que, aunque no hubo diferencias significativas entre los tratamientos probados para digerir el tejido calcificado, el tratamiento 5 mostró un porcentaje mayor de digestión y una menor dispersión de los datos (Figura 1). El uso de esta mezcla representa un ahorro de 50 % en el consumo del detergente y de 70 % en el consumo de proteinasa K, comparada con el protocolo de la desmineralización completa (Loreille, et al, 2007).…”

Section: Resultados Y Discusiónunclassified

“…Con base en los experimentos previos en el Laboratorio, así como en reportes de la literatura (Loreille, et al, 2007;Amory, et al, 2012), se partió de 1 g de material pulverizado para evaluar la eficiencia de la digestión, se utilizó un volumen de 15 ml de solución tampón para digestión compuesto de EDTA (JT Baker), lauril sarcosinato de sodio (LSS) (Sigma-Aldrich) y 10mg/ml de proteinasa K (pK) (AG Scientific).…”

Section: Materiales Y Métodosunclassified

“…Por ello, si se efectúa un proceso de desmineralización y se descarta el agente desmineralizante, se corre el riesgo de perder una buena parte del ADN. Es por eso que se han propuesto métodos de desmineralización y digestión simultáneos, con el fin de reducir la pérdida de ADN durante el proceso (Loreille, et al, 2007). Sin embargo, estas metodologías generalmente están ligadas a un solo método de purificación, que es el proceso siguiente e implica la separación de la molécula de interés del resto del material digerido.…”

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Protocolo unificado de digestión y descalcificación de tres métodos de extracción de ADN de restos humanos

Sierra¹,

del-Castillo-Sabogal²,

Espitia-Ortiz³

2018

Rev. Acad. Colomb. Cienc. Ex. Fis. Nat.

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ResumenLa identificación de restos humanos esqueletizados con base en el ADN es un procedimiento muy utilizado en los laboratorios forenses del mundo. Existen al menos tres métodos para la obtención del ADN a partir de este tipo de tejido: mediante solventes orgánicos (fenol-cloroformo), columnas de sílice (QIAquick de QIAgen) y perlas magnéticas recubiertas de polímero (PrepFiler Express™ BTA Forensic DNA Extraction Kit). Cada uno de ellos involucra un procedimiento distinto para la digestión y descalcificación del tejido óseo, así como la aplicación de diferentes principios fisicoquímicos para la purificación y la elución del ADN y el uso de diferentes cantidades iniciales de material pulverizado. Aunque los tres métodos han demostrado ser efectivos dependiendo de la calidad de la muestra, en el Laboratorio de Investigación Genética de Restos Humanos del Instituto Nacional de Medicina Legal y Ciencias Forenses se estandarizó un protocolo unificado para la digestión y descalcificación, independientemente del método de purificación. Con este fin, se evaluaron variables como la cantidad de material pulverizado, el uso y la concentración de detergentes, la concentración de proteinasa K, el uso de dispositivos concentradores y de los reactivos del estuche PrepFiler Express™ BTA Forensic DNA Extraction y EDTA. El efecto de las variables se evaluó en muestras de tejido calcificado provenientes de los casos forenses del Laboratorio y en el ADN de líneas celulares conocidas analizando la cantidad de ADN recuperado y la calidad del perfil genético. El protocolo unificado de digestión y decalcificación permite una digestión completa del material pulverizado y, por ende, una mejor recuperación de ADN, independientemente del método de purificación usado. En las muestras analizadas el método tuvo un porcentaje de éxito cercano a 80 %. © 2017. Acad. Colomb. Cienc. Ex. Fis. Nat. Palabras clave:Restos óseos; Digestión-decalcificación; Extracción de ADN; PrepFiler Express™; Sílice; Fenol-cloroformo. Abstract Unified digestion and decalcification protocol for three DNA extraction methods in human remainsThe identification of human remains using DNA from bones and teeth is a current procedure in forensic laboratories worldwide. At least three methods are used in DNA extraction from mineralized tissue: Organic (phenol-chloroform), silica (QIAquick, QIAgen) and magnetic particles (Prepfiler Express™ BTA Forensic DNA Extraction Kit); each one has its own procedure for decalcification, digestion, DNA purification, and elution, as well as for obtaining the initial amount of material. Although the three methods are equally efficient, we standardized a decalcificationdigestion protocol to use it with any of the three DNA purification methods. We evaluated the quantity of powder, detergent and protease concentration, device concentrators and buffer for demineralization in bones and teeth from forensic cases of the Laboratory and DNA of common cellular lines by assessing the amount of recovered DNA and the quality of the genetic profile. ...

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Section: Resultados Y Discusiónunclassified

Section: Materiales Y Métodosunclassified

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Protocolo unificado de digestión y descalcificación de tres métodos de extracción de ADN de restos humanos

Sierra¹,

del-Castillo-Sabogal²,

Espitia-Ortiz³

2018

Rev. Acad. Colomb. Cienc. Ex. Fis. Nat.

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“…For DNA extraction, samples of bones and teeth were subjected to demineralization, (Loreille et al, 2007) followed by centrifugation using the AMICON ULTRA filter (Millipore). Extraction procedures were carried out with the DNA IQ Kit (Promega) according to manufacturer instructions.…”

Section: Samplesmentioning

confidence: 99%

Analysis of SNP (single nucleotide polymorphism) multiplex markers related to sudden cardiac death in Brazilian families

Braganholi

Cicarelli

2015

Genet. Mol. Res.

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ABSTRACT. Sudden cardiac death (SCD) is a major public health concern worldwide, and genetic analysis may be useful in identifying the cause of death as well as in determining the possible genetic risk factors for SCD. This study analyzed eight SNPs (single nucleotide polymorphisms) highly correlated with cardiac sudden death in samples (blood and bone) from six Brazilian families with a history of cardiovascular diseases. Individuals with no family history of cardiovascular diseases were recruited as controls. Y chromosomes and mtDNA haplogroups belonging to these subjects were verified as well. We found that SNP rs16857031 showed significant differences between the group with a family history of cardiovascular diseases and the control group. Furthermore, the data obtained were compatible with known frequencies of SNPs for the haplogroups in which the samples were classified. A possible hereditary factor was identified for SNP rs4725982 in one family. These preliminary results suggest that identification of certain SNPs could be used to assess risk factors for SCD.

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“…Some studies indicate, that the fineness of the powder has influence on the quality of the DNA extracted, i.e. (19). Generally, we recommend dedicated instruments for aDNA only, however, in some laboratories, this is not feasible: Be aware of the contamination risks, apply precaution measures such as cleaning with chlorine bleach or similar.…”

Section: Physical Breakupmentioning

confidence: 99%

Ancient DNA and Forensics Mutual Benefits a Practical Sampling and Laboratory Guide Through a Virtual Ancient DNA Study

2014

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Genetic information discovered, characterized for and used in forensic case-works and anthropology has shown to be also highly useful and relevant in investigating human remains from archaeological findings. By technical means, forensic and aDNA (ancient Deoxyribonucleic acid) analyses are well suited to be done using the same laboratory infrastructures and scientific expertise referring to sampling, sample protection, sample processing, contamination control as well as requiring analogous technical know how and knowledge on reading and interpreting DNA encoded information. Forensic genetics has significantly profited from aDNA-related developments (and vice versa, of course!), especially, when it comes to the identification of unknown human remains referring to the detection limit. Additionally the tremendous developments of analyzing chemistry and kits as well as instruments in forensics opened the whole panel of reading human and nonhuman DNA for historians and archaeologists but also for anthropologists. Ancient DNA / molecular archaeology, however, is not limited to the comparatively restrictive set of information as usually employed in forensic case work analyses but can also be applied to phenotypical markers, ethno-related genotypes or pathological features.In this review the authors give a general overview on the field of ancient DNA analysis focussing of the potentials and limits, fields of application, requirements for samples, laboratory setup, reaction design and equipment as well as a brief outlook on current developments, future perspectives and potential cross links with associated scientific disciplines.Key words: Human DNA, Ancient DNA, Forensic DNA typing, Molecular archaeology, Application.

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High efficiency DNA extraction from bone by total demineralization

Cited by 252 publications

References 4 publications

Protocolo unificado de digestión y descalcificación de tres métodos de extracción de ADN de restos humanos

Protocolo unificado de digestión y descalcificación de tres métodos de extracción de ADN de restos humanos

Analysis of SNP (single nucleotide polymorphism) multiplex markers related to sudden cardiac death in Brazilian families

Ancient DNA and Forensics Mutual Benefits a Practical Sampling and Laboratory Guide Through a Virtual Ancient DNA Study

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