An efficient extraction method is important for obtaining high-quality DNA from degraded aged bone samples. An automated full-demineralization method using the EDTA and DNA Investigator Kit (Qiagen) ...combined with Qiagen’s biorobots was optimized in our laboratory in the past to extract the DNA from 500 mg of aged bone samples. The purpose of this research was to further improve the method with the aim of reducing the required sample material, shortening the extraction time, and achieving higher throughput. To process extremely small samples, the amount of bone powder was reduced to 75 mg, EDTA was replaced with reagents from the Bone DNA Extraction Kit (Promega), and decalcification was shortened from overnight to 2.5 h. Instead of 50 ml tubes, 2 ml tubes were used, which allows higher throughput. The DNA Investigator Kit (Qiagen) and EZ1 Advanced XL biorobot (Qiagen) was used for DNA purification. A comparison between both extraction methods was made on 29 Second World War bones and 22 archaeological bone samples. The differences between both methods were explored by measuring nuclear DNA yield and STR typing success. After cleaning the samples, 500 mg of bone powder was processed using EDTA, and 75 mg of powder from the same bone was processed using the Bone DNA Extraction Kit (Promega). DNA content and DNA degradation were determined using PowerQuant (Promega), and the PowerPlex ESI 17 Fast System (Promega) was used for STR typing. The results showed that the full-demineralization protocol using 500 mg of bone was efficient for Second World War and archaeological samples, and the partial-demineralization protocol using 75 mg of bone powder was only efficient for the Second World War bones. The improved extraction method—for which significantly lower amounts of bone powder can be used, the extraction process is faster, and higher throughput of bone samples is possible—is applicable for genetic identification of relatively well-preserved aged bone samples in routine forensic analyses.
•Identification of small bone samples requires an efficient DNA extraction method.•Full demineralization of 500 mg of bone and partial demineralization of 75 mg were compared.•WWII and archaeological bones were used for analysis.•The full-demineralization method was efficient for WWII and archaeological bones.•The partial-demineralization method was efficient only for WWII bones.
The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA ...community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille's overall increased gain of DNA when enough tissue is available and Dabney's improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.
Abstract DNA genotyping techniques have been used successfully in forensic science for almost three decades and represent the gold standard for individual identification. However, efficient protocols ...for obtaining DNA from exhumed bones suitable for genotyping are still scarce and most of them require a considerable amount of starting material, are time consuming and are inefficient for reducing inhibitor's effects. We sought to develop an optimised protocol for extracting DNA from bone samples obtained from exhumations. We tested two approaches for preparing bone samples: (a) fine powder and (b) thin slices of bone. The best ratio of bone amount to DNA yields was assessed by a titration experiment using bone powder ranging from 50 to 1000 mg. We obtained optimal DNA yields (27 pg mg−1 on average) when 150–200 mg of starting material were processed using a one-step demineralisation method. Better-quality profiles (determined by the number of genotyped loci) were obtained when DNA was extracted from bone slices compared to extraction from bone powder. From bone slices 83.9% and from bone powder 46.7% of the samples provided genotypes for 11 or more loci. Since bone preparation procedures were carried out at room temperature, the method developed in the present study might be an attractive alternative to the standard freeze-mill approach, being faster and more cost-efficient.
Deoxyribonucleic acid (DNA) sequence analysis of the control region of the mitochondrial DNA (mtDNA) genome was used to identify human skeletal remains returned to the United States government by the ...Vietnamese government in 1984. The postmortem interval was thought to be 24 years at the time of testing, and the remains presumed to be an American service member. DNA typing methods using nuclear genomic DNA, HLA-DQ alpha and the variable number of tandem repeat (VNTR) locus D1S80, were unsuccessful using the polymerase chain reaction (PCR). Amplification of a portion of the mtDNA control region was performed, and the resulting PCR product subjected to DNA sequence analysis. The DNA sequence generated from the skeletal remains was identical to the maternal reference sequence, as well as the sequence generated from two siblings (sisters). The sequence was unique when compared to more than 650 DNA sequences found both in the literature and provided by personal communications. The individual sequence polymorphisms were present in only 23 of the more than 1300 nucleotide positions analyzed. These results support the observation that in cases where conventional DNA typing is unavailable, mtDNA sequencing can be used for human remains identification.
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