Elsevier

Legal Medicine

Volume 22, September 2016, Pages 36-41
Legal Medicine

Comparison of two silica-based extraction methods for DNA isolation from bones

https://doi.org/10.1016/j.legalmed.2016.07.008Get rights and content

Highlights

  • Two silica based DNA extraction methods using silica beads or silica membranes were compared.

  • Testing of bone samples of various qualities with both methods show remarkable differences in DNA yield and purity.

  • Bead-technology results in excellent purity but the membrane-based procedure yields significantly more DNA.

  • The right choice of the extraction method depending on the quality of the bone sample is important.

Abstract

One of the most demanding DNA extractions is from bones and teeth due to the robustness of the material and the relatively low DNA content. The greatest challenge is due to the manifold nature of the material, which is defined by various factors, including age, storage, environmental conditions, and contamination with inhibitors. However, most published protocols do not distinguish between different types or qualities of bone material, but are described as being generally applicable. Our laboratory works with two different extraction methods based on silica membranes or the use of silica beads. We compared the amplification success of the two methods from bone samples with different qualities and in the presence of inhibitors. We found that the DNA extraction using the silica membrane method results an in higher DNA yield but also in a higher risk of co-extracting impurities, which can act as inhibitors. In contrast the silica beads method shows decreased co-extraction of inhibitors but also less DNA yield. Related to our own experiences it has to be considered that each bone material should be reviewed independently regarding the analysis and extraction method. Therefore, the most ambitious task is determining the quality of the bone material, which requires substantial experience.

Introduction

One of the most challenging DNA extractions is from bones and teeth, as the material is robust and has generally low DNA content. However, the greatest challenge is generally the manifold nature of the material, which is defined by various factors, including age, storage, bacterial and environmental degradation, and contamination by inhibitors such as fulvic and humic acids, tannins, phenolic compounds, or complexing ions (e.g., iron) [1], [2], [3]. Perhaps due to the variety of bone materials, a number of different extraction protocols are used, and they are difficult to compare regarding their efficiency. Only a few improvements or guidelines have been made in the past. For example, total decalcification with higher EDTA concentrations is more efficient than incomplete decalcification [4], [5]. A comparison of phenol/chloroform (organic) or silica-based methods for DNA extraction has shown that silica-based technology has better DNA recovery and more efficient removal of inhibitors [6], [7], [8]. However, even when considering only the silica-based extraction method there is high variation. For example, many protocols use columns with a silica membrane, which are included in various extraction or purification kits [6], [9], [10]. Other protocols rely on the use of silica beads [11], [12], and yet others use a silica suspension [13]. Basically, all protocols differ in the use of decalcification and digestion buffer, incubation temperature, and time. Most of the published protocols do not distinguish between different types or quality of bone materials but are described as being generally applicable. Only a few protocols refer to a specific type of sample, such as the use of size exclusion chromatography for the removal of metal ions from copper- or bronze-contaminated samples [14]. Relating to our own experiences, each material needs to be considered on its own, with its own adapted form of analysis, and sometimes unexpected challenges can occur that can hardly be generalized. Therefore, we think that the exchange of data and experience is vitally important in the field of DNA extraction from bones and teeth. Here, we compare two different protocols for DNA extraction from bones and teeth, as well as their advantages and disadvantages.

Section snippets

Laboratory quality assurance and bone samples

Taking commonly applied precautions (e.g. Hummel, 2003 [15], Pääbo et al., 2004 [16]) all pre-PCR work was performed in laboratory facilities exclusively dedicated to investigations of skeletal remains. Detailed description of the techniques to avoid contamination with exogenous DNA, the criteria of authenticity and sample preparation are explained in Rothe et al. [17].

Bone samples were taken from different case work with forensic, archaeological or ancestry relevant background and showed

Comparison of DNA yield between DNA extraction with silica membrane and silica beads

In this study we analyzed a relatively broad range of different sample qualities (Table 1), which primarily differed in exposure times. The oldest (ancient) samples were specimens 1 and 2 with an exposure time of more than 500 years. Specimens 3, 4, and 5 had a more moderate age of approximately 50–90 years. Specimens 6 and 7 were of recent age, whereas specimen 8 was derived from fresh bone. However, in addition to the exposure time, other specimen characteristics influenced the bone quality.

Discussion

In this study, we compared two commercially available extraction methods. First, the MinElute PCR Purification kit, which uses a silica membrane in combination with a chaotropic salt-containing binding buffer. Second, the EZ1® Investigator kit, which works with silica-covered magnetic beads in combination with a chaotropic salt-containing binding buffer. In both methods, DNA is absorbed by a silica surface in the presence of a high concentration of salt and a pH  7.5. Although both methods are

Conclusions

In summary, we cannot state that either of the investigated extraction methods is the best one, but both have their advantages and disadvantages. Although these two methods are based on the same chemistry, we have found strong variations in the DNA yield and co-extraction of inhibitors. The method of choice should be decided by the quality of the samples. For example, the beads method should be the choice for more recent samples or samples with high amounts of inhibitors, whereas samples with

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