Developmental Validation of the Bone DNA Extraction Kit

Jonelle M Thompson, Arune K Brekke, Margaret M Ewing, Anupama Gopalakrishnan, Douglas Horejsh | Promega Corporation, 2800 Woods Hollow Rd., Madison, WI 53711

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Introduction

The extraction of DNA from bone and teeth samples presents unique challenges due to their complex and dense matrix composition. Traditional lysis reagents used for extracting DNA from other biological samples are often ineffective when applied to bone, necessitating specialized protocols to achieve efficient DNA recovery. The Bone DNA Extraction Kit addresses these challenges by utilizing a Demineralization Buffer protocol, originally formulated by the Armed Forces DNA Identification Laboratory, which has proven effective across various purification methods (1,2).

This kit includes all necessary reagents for the preprocessing of pulverized bone samples and offers flexibility with both manual and automated DNA purification options. The Demineralization Buffer included in the kit is composed of thylenediaminetetraacetic acid (EDTA) and 1% lauroylsarcosine. The EDTA chelates calcium and bivalent enzymatic cofactors, dissolving the bone matrix and inactivating DNases. The lauroylsarcosine acts as a detergent, enabling cell lysis and protein denaturation (2). The integration of DNA IQ™ chemistry ensures that the extraction process can be seamlessly adapted for use with either manual techniques or automated platforms like the Maxwell® Instrument, thereby providing a robust solution for forensic laboratories.

The primary objective of this extraction method is to maximize the yield and quality of DNA recovered from challenging bone samples, facilitating downstream applications such as human DNA quantification and short tandem repeat (STR) analysis. The formulation of the Bone DNA Extraction Kit reagents is identical to the formulation for Bone DNA Extraction Kit Custom, reagents. Studies evaluating the performance of the custom kit with challenging samples have been described in literature (3,4). By enhancing DNA recovery and enabling automated purification on a Maxwell® instrument, the Bone DNA Extraction Kit boosts the efficiency and reliability of forensic DNA analysis of bone samples.

This report details the validation and performance of the Bone DNA Extraction Kit, highlighting its efficiency and reliability in processing bone and teeth samples. These studies are based on requirements listed in the FBI Quality Assurance Standards for Forensic DNA Testing Laboratories (5) and guidelines outlined by the Scientific Working Group on DNA Analysis Methods6. Through validation studies, this protocol has demonstrated its capability to produce reliable extracts for use in the forensic laboratory.

Materials and Methods

Samples

Two human femur bone samples were purchased from BioIVT (NY, USA). Six tooth samples were collected from donors following Promega’s informed consent protocol.

DNA Extraction and Purification

Sample preprocessing was performed using the Bone DNA Extraction Kit in accordance with the protocol in the technical manual (2). Four hundred microliters of bone lysis cocktail A (Table 1) were combined with 100 mg of pulverized bone or tooth powder, unless otherwise specified.

Each sample was incubated at 56°C for 2.5 hours while shaking at 1,000 rpm. After incubation, each sample was centrifuged for 5 minutes at 13,000 x g. The supernatant was then transferred and combined with 800 µL of bone lysis cocktail B (Table 2).

The resulting lysate was purified using the Maxwell® FSC DNA IQ™ Casework Kit on the Maxwell® RSC 48 instrument (7). The entire lysate for each sample was transferred to well 1 of the Maxwell® FSC cartridge where it was mixed using a pipette tip. After mixing, the DNA IQ™ Casework protocol was run. The elution volume was 50 µL.

Table 1: Composition of Bone Lysis Cocktail A

Table 2: Composition of Bone Lysis Cocktail B

DNA Quantification

The extracted DNA from each sample was quantified using the PowerQuant® System (Promega Corp, Madison, WI) on the Applied Biosystems 7500 Real-Time PCR System (Waltham, MA) following the technical manual protocol (8). The following sample quality flags were monitored throughout the studies: the autosomal to degradation ratio ([Auto]/[Deg]), the total human to male ratio ([Auto]/[Y]) and IPC (internal positive control) Cq shift. These quality flags provide information about the sample and probable STR profile quality. A threshold of 2 was used for the [Auto]/[Deg] and [Auto]/[Y] ratios to flag a sample as possibly degraded or as a potential mixture, respectively. A shift of 0.3 for the IPC Cq was used to flag a sample for possible inhibition.

STR Analysis

DNA from all tooth samples and each bone was amplified with the PowerPlex® 18E System9 and PowerPlex® 35GY System10 (Promega Corp, Madison, WI) following the protocol detailed in their respective technical manuals. A total of 0.5 ng or up to 15 μL of sample was added to each PowerPlex® 18E System amplification reaction. A total of 1ng or up to 15 μL of sample was added per to each PowerPlex® 35GY System amplification reaction. Samples were amplified using the ProFlex™ PCR System (Applied Biosystems, Waltham, MA), and 1 μl of amplified product was used for electrophoresis. All samples were injected at 2 kV for 15 seconds and run using the Promega 8-Dye (8C) protocol11,12. Files generated by the Spectrum CE Systems (.promega format) were analyzed with GeneMarker® HID Software for Spectrum CE Systems13 (v3.2.0) (Promega Corp, Madison, WI).

Sensitivity Study

A sensitivity study was performed to evaluate the linear range of DNA extracted. The following input amounts were tested for two different bone donors (B1 and B2): 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, and 200 mg. Each input amount was processed in triplicate. B1 is a female sample, while B2 is a male sample.

Precision Study

The precision of DNA quantity and quality obtained from replicate samples was assessed. DNA from 100 mg of bone powder was extracted in triplicate. The extraction setup was performed twice to compare replicates within and between extraction sets.

Contamination Study

To assess sample-to-sample contamination, extraction reagent blanks were processed alongside bone and tooth samples within each extraction set. This approach ensured that any potential cross-contamination between samples could be identified and accounted for during the analysis.

Known and Casework Samples

Six tooth samples were tested from known donors. The collection dates varied between the samples collected (Table 3).

Table 3: Description of tooth samples

Bone Lysis Cocktail B Volume Study

The volume of the bone lysis cocktail B was adjusted to evaluate the impact of volume changes on the quantity of DNA obtained. The following input amounts from one donor were tested: 50 mg, 100 mg, and 150 mg. Each of these input amounts was extracted using the following bone lysis cocktail B volumes: 200 µL, 400 µL, 600 µL, and 800 µL. Each combination was extracted in triplicate.

Results and Discussion

Sensitivity Study

Quantifiable levels of DNA were detected from all extracted samples in the sensitivity study. The average concentrations obtained from B1 ranged from 34.6 ng/µL for 25 mg samples to 50.5 ng/µL from 200 mg samples (Figure 1) and showed linear increases up to 100 mg (Figure 2).

Figure 3 shows a representative PowerPlex® 18E System electropherogram from 0.5 ng of template DNA. The average concentrations obtained from B2 ranged from 4.1 ng/µL for 25 mg samples to 21.2 ng/µL for 200 mg samples (Figure 4) and showed linear increases through the entire sensitivity series (Figure 5).

Figure 6 shows a representative PowerPlex® 35GY electropherogram from 1ng of template DNA. There were no IPC shift flags.

Figure 1: DNA concentration from each extraction for B1. The X axis represents the input amount of bone powder used for sample extraction. The Y axis represents the average DNA concentration (Autosomal and Degradation targets). The error bars represent ±1 standard deviation. n=3.

Figure 2: Linearity of DNA concentration from each extraction for B1. The X axis represents the input amount of bone powder used for sample extraction. The Y axis represents the average DNA concentration (Autosomal target). The error bars represent ±1 standard deviation. n=3.

Figure 3: Representative DNA profile of B1. The sample was extracted using the Bone DNA Extraction kit and purified on the Maxwell® RSC 48 Instrument with the Maxwell® FSC DNA IQ™ Casework Kit and quantified using the PowerQuant® System on the Applied Biosystems 7500 Real-Time PCR System. Amplification was performed using the PowerPlex® 18E System on a ProFlex™ PCR System thermal cycler and electrophoresed on a Spectrum CE System. [Auto]/[Deg] 2.59.

Figure 4: DNA concentration from each extraction for B2. The X axis represents the input amount of bone powder used for sample extraction. The Y axis represents the average DNA concentration (Autosomal, Y, and Degradation targets). The error bars represent ±1 standard deviation. n=3.

Figure 5: Linearity of DNA concentration from each extraction for B2. The X axis represents the input amount of bone powder used for sample extraction. The Y axis represents the average DNA concentration (Autosomal target). The error bars represent ±1 standard deviation. n=3.

Figure 6: Representative DNA profile of B2. This sample was extracted using the Bone DNA Extraction kit and purified on the Maxwell® RSC 48 Instrument with the Maxwell® FSC DNA IQ™ Casework Kit and quantified using the PowerQuant® System on the Applied Biosystems 7500 Real-Time PCR System. Amplification was performed using the PowerPlex® 35GY System on a ProFlex™ PCR System thermal cycler and electrophoresed on a Spectrum CE System. [Auto]/[Deg] 4.4.

Precision Study

The variability within extraction runs and between extraction runs ranged from 3 to 11% (Table 4). IPC Cq flags were not detected with the PowerQuant® System in any sample.

Table 4: Average concentration, standard deviation (St.Dev), and coefficient of variation (CV%) across PowerQuant® System targets for replicates within runs and between runs. Blue cells represent Run 1, green cells represent Run 2, and yellow cells represent calculations between runs.

Contamination Assessment

Six extraction reagent blanks were assessed throughout the validation process. DNA was detected in one of the samples using the PowerQuant® System. When amplified with both the PowerPlex® 18E and PowerPlex® 35GY Systems, one peak at 210 RFU was present in PowerPlex® 18E (data not shown) while no peaks were present in PowerPlex® 35GY. No other indications of contamination were observed in the profile.

Known and Casework Samples

Average autosomal DNA concentrations ranged between 0.0098 ng/µL to 0.5138 ng/µL for the tooth samples (Table 5). No IPC Cq flags were detected with the PowerQuant® System, while all samples except casework 4 had flags for [Auto]/[Deg]. The QIS/QIL ratio in the STR profiles were balanced, ranging from 0.67 to 1. This suggests that inhibitors were not a factor in peak height, and any decrease in peak height at larger loci is likely due to degradation rather than inhibition.

Table 5: Casework sample quantification results.

The quantification and STR results for casework 6 were consistent with a male and female mixture. This mixture was expected due to combining teeth from two donors during the grinding process (Figure 7).

Examples of STR profiles are provided for samples with different levels of degradation (Figures 8 and 9).

Figure 7: Mixed DNA profile of sample Casework 6. Sample was extracted using the Bone DNA Extraction kit and purified on the Maxwell® RSC 48 Instrument with the Maxwell® FSC DNA IQ™ Casework Kit and quantified using the PowerQuant® System on the Applied Biosystems 7500 Real-Time PCR System. Amplification was performed using the PowerPlex® 18E System on a ProFlex™ PCR System thermal cycler, and electrophoresed on a Spectrum CE System. [Auto]/[Deg] UND, [Auto]/[Y] 7.

Figure 8: Single source male DNA profile of sample Casework 4. Sample was extracted using the Bone DNA Extraction kit and purified on the Maxwell® RSC 48 Instrument with the Maxwell® FSC DNA IQ™ Casework Kit and quantified using the PowerQuant® System on the Applied Biosystems 7500 Real-Time PCR System. Amplification was performed using the PowerPlex® 35GY System on a ProFlex™ PCR System thermal cycler and electrophoresed on a Spectrum CE System. [Auto]/[Deg] 1.11, [Auto]/[Y] 1.36

Figure 9: Single source female DNA profile for Casework 1. Sample was extracted using the Bone DNA Extraction kit and purified on the Maxwell® RSC 48 Instrument with the Maxwell® FSC DNA IQ™ Casework Kit and quantified using the PowerQuant® System on the Applied Biosystems 7500 Real-Time PCR System. Amplification was performed using the PowerPlex® 18E System on a ProFlex™ PCR System thermal cycler, and electrophoresed on a Spectrum CE System. [Auto]/[Deg] 9.17.

Bone Lysis Cocktail B Volume Study

The concentration of DNA increased when the volume of bone lysis buffer cocktail B was increased, except for the 150 mg sample when comparing the 600 µL and 800 µL samples (Figure 10). Samples that were extracted with 200 µL had the lowest concentration for all input amounts tested.

Figure 10: DNA concentration from each extraction for B1 with varying volumes of Bone Lysis cocktail B. The X axis represents the input amount of bone powder used for sample extraction. The Y axis represents the average DNA concentration (Autosomal target). Bar colors represent differences in the volume of Bone Lysis Buffer Component B (dark blue = 200 µL, orange = 400 µL, green = 600 µL, and light blue = 800 µL). The error bars represent ±1 standard deviation.

Conclusion

A series of validation studies were conducted to evaluate the performance of the Bone DNA Extraction Kit when used with the Maxwell® RSC 48 Instrument, in conjunction with the Maxwell® FSC DNA IQ™ Casework Kit. These studies aimed to assess the kit’s reliability and suitability for forensic applications. The results demonstrate the system’s effectiveness in reliably generating high quality DNA extracts from bone and tooth samples. The validation demonstrates that the Bone DNA Extraction Kit is suitable for use in a forensic laboratory.

Acknowledgements

The authors would like to acknowledge Sarah Bettinger for her contributions to this effort.

References

  1. Lorielle, O.M. et al. (2007) High efficiency DNA extraction from bone by total demineralization. Forensic Sci. Int. Genet. 1, 191–5.
  2. Bone DNA Extraction Kit Technical Manual TM691, Revision Date 8/22 from Promega Corporation; available at https://www.promega.com/-/media/files/resources/protocols/technical-manuals/500/bone-dna-extraction-kit-protocol-tm691.pdf?sc_lang=en?la=en
  3. Calacal, G.C. et al. (2021) Improved autosomal STR typing of degraded femur samples extracted using a custom demineralization buffer and DNA IQ™. Forensic Sci. Int. Synerg. 3, 100131. 3.
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  5. Federal Bureau of Investigation (2020) Quality Assurance Standards for Forensic DNA Testing Laboratories; available at 2020 Quality Assurance Standards for Forensic DNA Testing Laboratories (swgdam.org)
  6. Scientific Working Group on DNA Analysis Methods (2016) Validation Guidelines for DNA Analysis Methods; available at https://www.swgdam.org/publications
  7. Maxwell® FSC DNA IQ™ Casework Kit Technical Manual TM499, Revision Date 1/24 from Promega Corporation; available at https://www.promega.com/resources/protocols/technical-manuals/101/maxwell-fsc-dna-iq-casework-kit-protocol/
  8. PowerQuant® System Technical Manual TMD047, Revision Date 08/22 from Promega Corporation; available at https://www.promega.com/resources/protocols/technical-manuals/d0/powerquant-system-protocol/
  9. PowerPlex® 18E System for Use on the Spectrum CE System Technical Manual TMD080, Revision Date 4/24 from Promega Corporation; available at https://www.promega.com/resources/protocols/technical-manuals/d0/powerplex-18e-system-spectrum-ce-system-protocol/
  10. PowerPlex® 35GY System for Use on the Spectrum CE System Technical Manual TMD077, Revision Date 7/23 from Promega Corporation; available at https://www.promega.com/resources/protocols/technical-manuals/d0/powerplex-35gy-system-for-use-on-the-spectrum-ce-system-protocol/
  11. Promega Corporation. Spectrum CE System Operating Manual #TMD052. Madison, WI; revised 4/24. Available from: https://www.promega.com/resources/protocols/technical-manuals/d0/spectrum-ce-system-operating-manual/
  12. Promega Corporation. PowerPlex® Matrix Standards for Use on the Spectrum CE System Technical Manual #TMD068. Madison, WI; revised 1/23. Available from: https://www.promega.com/resources/protocols/technical-manuals/d0/powerplex-matrix-standards-spectrum-ce-system-protocol/
  13. Promega Corporation. GeneMarker® HID Software for Spectrum CE Systems User Manual #TM555. Madison, WI; revised 1/23. Available from: https://www.promega.com/resources/protocols/technical-manuals/500/genemarker-hid-software-for-spectrum-ce-systems-user-manual/

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