Evaluation of techniques for human bone decalcification and amplification using sixteen STR markers

Balayan, Ajay; Kapoor, Abhilasha; Chaudhary, Garima; Raina, Anupuma

doi:10.1016/j.ejfs.2014.05.002

Cited by 7 publications

(8 citation statements)

References 5 publications

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“…Skeletal remains are often the only source of DNA for identifying a human body. Some studies have been performed to evaluate techniques for bone/tooth decalcification and their effects on DNA extraction [ 16 , 17 ]. The purpose of decalcification is to destroy the inorganic phase by removing calcium while avoiding damage to the organic phase.…”

Section: Resultsmentioning

confidence: 99%

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Barbaro

Samar

Falcone

et al. 2021

Forensic Sciences Research

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Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth. KEY POINTSBones and teeth often represent the only sources of DNA for identifying human remains. The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors. This study focuses on modifications to the previously available Qiagen-based protocols. The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols. The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.

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Section: Resultsmentioning

confidence: 99%

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Barbaro

Samar

Falcone

et al. 2021

Forensic Sciences Research

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“…Previous publications have reported the suitable and applicable method for bone preparation before DNA extraction such as demineralization. This process is based on the use of EDTA to chelate some mineral composition in specimens (Balayan et al 2015). The concentration and pH of EDTA were previously reported to affect the demineralization process of the teeth or on the bovine cortical bone (Sales et al 2018).…”

Section: Discussionmentioning

confidence: 99%

“…Our result was concordance with previous reports observing in old sternum human bone (more than 20 years of preservation). DNA profiling was improved when using a high concentration of EDTA (0.5 M) in the demineralization process (Balayan et al 2015). The comparison of different concentrations of EDTA revealed that the EDTA concentration of 0.5 M was capable to chelate the highest amount of calcium and phosphorus from the bones and teeth (Serper and Çalt 2002;Sales et al 2018).…”

Section: Discussionmentioning

confidence: 99%

“…3 Amplification of a 113-bp fragment on 1.5% agarose gel. Lane marker, molecular weight marker and lanes 0-13, and 4 μl of bone from different demoralization process (from experimental groups 0, 1, 2, 4, 8, 9, and 13) were used as a template might improve STR profiles in skeletal remains as described elsewhere (Bender et al 2000;Loreille et al 2007;Bilic et al 2012;Jakubowska et al 2012;Balayan et al 2015;Zupanič Pajnič et al 2016). Therefore, the demineralization process according to experimental group no.…”

Section: Discussionmentioning

confidence: 99%

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Factors influencing DNA extraction from human skeletal remains: bone characteristic and total demineralization process

Booncharoen

Khacha-ananda

Kanchai

et al. 2021

Egypt J Forensic Sci

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Background The extraction of DNA from skeletal remains with good quality and quantity is often challenging for the ability to generate DNA typing. Previous studies demonstrated the DNA extraction with total demineralization from fresh teeth and bones; however, the application in old skeletal remains has been less performed. To obtain good quality and high yield of DNA amount extracted from skeletal remains, the objective of this study was focused on exploring the factors influencing the total demineralization process to obtain developing effective methods. Results The concentration of EDTA was found to significantly enhance calcium chelation from the bone while pH of EDTA solution, incubation temperature, incubation time, and volume of EDTA solution were not significant. The optimal condition of total demineralization obtained from Placket-Burmann results represented good-quality DNA and the highest concentration of extracted DNA yield. Subsequently, the STR typing in some bone specimens processed by total demineralization process prior to DNA extraction was improved. Conclusions EDTA concentration was a key influencing factor on the total demineralization process to chelate calcium from human skeletal remains. The total demineralization process in old bone specimens probably improved the STR profiles.

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“…Descaling is based on inorganic phase destruction of bone tissue, to calcium removal, that will get to the organic portion of the bone. To this end, the techniques more usually employed are those, which use strong acids, which form soluble calcium salts and chelating agents, who kidnap the calcium ions leaving the organic portion more pliable and easy removal [4].…”

Section: Introductionmentioning

confidence: 99%

Alternative methodology for extraction of high-quality DNA from ancient bones by demineralization without pulverization

Silva¹,

Ekert²,

Mazanek³

et al. 2018

Forensic Sci Criminol

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Introduction:The robustness of the DNA molecule, coupled with the fact that it contains digital information, makes it ideal as a source of identification which resists weather and environmental aggressions imposed on samples found in crime scenes, but these same factors make these samples exhibiting low DNA concentration and with degradation in your chemical structure. The recovery of remaining genetic material of human skeletons is essential part human forensic identification, because the genetic identification by DNA is a method that outperforms all other existing methods.Objective: This work aims to obtain DNA from bone samples degraded and produce an electropherogram with the minimum number of markers recommended by the scientific community forensic, using a simple and effective protocol. Materials and methods:In order to analyse the DNA, samples of the bones and the teeth was demineralized in an EDTA solution and extracted using the Microcon® tube protocol. PCR analysis system approached by STRs PowerPlex Fusion 6C® system, PowerPlex Fusion® system and Globalfiler® System and Capillary electrophoresis procedures were performed on the ABI 3500 Genetic Analyzer. Results:The DNA extracted from the teeth sample and from the bone sample with demineralization without pulverization presented a male genetic profile complete with all amplification system markers. Conclusions:Our alternative extraction protocol proved to be efficient because it allowed the isolation of DNA from high quality of a 17-year bone buried, in order to present a complete genetic profile of all system markers.

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Evaluation of techniques for human bone decalcification and amplification using sixteen STR markers

Cited by 7 publications

References 5 publications

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Factors influencing DNA extraction from human skeletal remains: bone characteristic and total demineralization process

Alternative methodology for extraction of high-quality DNA from ancient bones by demineralization without pulverization

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