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

Booncharoen, Pichittra; Khacha-ananda, Supakit; Kanchai, Chaturong; Ruengdit, Sittiporn

doi:10.1186/s41935-021-00216-8

Cited by 6 publications

(5 citation statements)

References 27 publications

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“…The following morning the powder was centrifuged for 5 min at 2000 × g at room temperature, the EDTA was discarded, and the pellet was washed twice with TE‐4 (10 mM Tris‐Cl, pH 8.0 + 0.1 mM EDTA) by resuspension and centrifugation. There are several publications that describe demineralization of bone using EDTA [8, 14]. In the protocol used here, residual bone powder often remained after soaking in 0.5 M EDTA.…”

Section: Methodsmentioning

confidence: 99%

“…There are several publications that describe demineralization of bone using EDTA [8,14]. In the protocol used here, residual bone powder often remained after soaking in 0.5 M EDTA.…”

Section: Preparation Of Human Remains For Dna Extractionmentioning

confidence: 99%

“…Extraction workflows often begin with demineralization of the bone powder during an incubation with EDTA [13][14][15] followed by cellular disruption with proteinase K/SDS digestion or high concentrations of chaotropic salt [4,[8][9][10][11][12][13] and gDNA was generally recovered by binding to silica [12,13].…”

Section: Highlightsmentioning

confidence: 99%

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An analysis of data curated from 5 years of identifying human remains

Allen

Pritchard

Jun

2023

Journal of Forensic Sciences

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An archive of 5 years of cases involving the identification of human remains was curated, collecting information on: The sample type submitted, the number of STR loci yielding interpretable results, the kinship challenge posed, and the outcome for the case. A total of 129 cases of remains ID were investigated using manual DNA extraction and recovery methods with amplification of STR markers using the Power Plex 21 multiplex STR kit from Promega Corp. In 52 cases, blood spots collected by the ME were provided as sample and in 100% of those cases, probabilities of relatedness to the reference samples was ≥99%. In 77 cases, tissue other than blood was provided as a source of DNA. These other samples were grouped categorically into long bones (femur and tibia; 40 cases), skull bones/teeth (11 cases), other bones (16 cases), and tissue (normally adherent to bone) (10 cases). Reference samples provided for cases included alleged parents or child(ren) of the victim (86 cases), alleged full siblings of the victim (38 cases), or alleged second‐order relatives (five cases). The overall success rate in confirming the identity of the source of the remains in these cases was 89.2%. Our results demonstrate that a laboratory can be often successful identifying human remains using methods easily implemented in any DNA typing laboratory.

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Section: Methodsmentioning

confidence: 99%

“…There are several publications that describe demineralization of bone using EDTA [8,14]. In the protocol used here, residual bone powder often remained after soaking in 0.5 M EDTA.…”

Section: Preparation Of Human Remains For Dna Extractionmentioning

confidence: 99%

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An analysis of data curated from 5 years of identifying human remains

Allen

Pritchard

Jun

2023

Journal of Forensic Sciences

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“…Optimization of methods and protocols often requires evaluation of numerous factors that might impact performance, and tools for statistical design of experiments can assist this process by minimizing the number of required experiments. Booncharoen et al (2021) evaluated factors that can influence DNA extraction from bones such as the concentration of the chelator (EDTA), pH, incubation time and temperature, and volume of the solution. To fully explore the combined effect of all these factors, they used the Plackett-Burmann statistical method to design the experimental approach and the number of experiments for protocol optimization was reduced from 32 to 13, and enabled them to identify EDTA concentration as the major factor of success for the protocol ( Booncharoen et al, 2021 ).…”

Section: Generation and Analysis Of Genomics Data For The Study Of Mi...mentioning

confidence: 99%

Advances in experimental and computational methodologies for the study of microbial-surface interactions at different omics levels

et al. 2022

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The study of the biological response of microbial cells interacting with natural and synthetic interfaces has acquired a new dimension with the development and constant progress of advanced omics technologies. New methods allow the isolation and analysis of nucleic acids, proteins and metabolites from complex samples, of interest in diverse research areas, such as materials sciences, biomedical sciences, forensic sciences, biotechnology and archeology, among others. The study of the bacterial recognition and response to surface contact or the diagnosis and evolution of ancient pathogens contained in archeological tissues require, in many cases, the availability of specialized methods and tools. The current review describes advances in in vitro and in silico approaches to tackle existing challenges (e.g., low-quality sample, low amount, presence of inhibitors, chelators, etc.) in the isolation of high-quality samples and in the analysis of microbial cells at genomic, transcriptomic, proteomic and metabolomic levels, when present in complex interfaces. From the experimental point of view, tailored manual and automatized methodologies, commercial and in-house developed protocols, are described. The computational level focuses on the discussion of novel tools and approaches designed to solve associated issues, such as sample contamination, low quality reads, low coverage, etc. Finally, approaches to obtain a systems level understanding of these complex interactions by integrating multi omics datasets are presented.

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“…To obtain results from small DNA fragments, an extraction method able to obtain a high yield of DNA from such degraded and low-copy samples has become critical and the limiting factor. Bone and rootless hairs are considered "challenging" samples in forensics because they tend to contain low quantities of highly degraded nuclear DNA, mainly when the sample is aged or has been subjected to chemical and/or physical insults [3][4][5][6][7][8][9][10][11] The problem of extracting DNA from degraded bone and hair shaft samples is compounded by the fact that the quality and quantity of DNA recoverable from bone [12][13][14][15] and hair shafts [16] varies widely in the same individual and even in different locations on the same sample. These samples also contain PCR inhibitors, such as melanin in hair and calcium and humic acid in bone, which disrupt downstream quantification and amplification processes [17][18][19].…”

Section: Introductionmentioning

confidence: 99%

Validation of the InnoXtract™ DNA extraction method for bone, teeth, and rootless hair

Sinha

Brown

Holt

et al. 2023

Journal of Forensic Sciences

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Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high‐yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non‐probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un‐insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express‐96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high‐throughput, high‐yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high‐yield and high‐quality DNA from compromised bone, teeth, and hair shaft samples.

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

Cited by 6 publications

References 27 publications

An analysis of data curated from 5 years of identifying human remains

An analysis of data curated from 5 years of identifying human remains

Advances in experimental and computational methodologies for the study of microbial-surface interactions at different omics levels

Validation of the InnoXtract™ DNA extraction method for bone, teeth, and rootless hair

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