Long-Term DNA Storage of Challenging Forensic Casework Samples at Room Temperature

Abstract

Background: The success of forensic genetics has led to considerable numbers of DNA samples that must be stored. For example, the genetic casework unit of the forensic institute of the French gendarmerie analyzes more than 70,000 casework samples per year mainly from swabs that are fully consumed during DNA extraction. The only way to process further analyses is to preserve DNA. Currently, the most common technique used for the long-term preservation of DNA is to freeze the extracted DNA at −20 °C or −80 °C. However, this preservation method involves significant constraints (large equipment), risks (equipment failure), and is not ecologically sustainable due to its high energy consumption. Many solutions for DNA preservation at room temperature exist based either on fibrous supports or on anhydrobiosis. However, few studies have examined the efficiency of these systems in preserving very-low DNA amounts, such as those in forensic samples (≤1 ng), while ensuring full recovery and the ability to retest the samples many years later. Methods: We choose to evaluate the ability of the anhydrobiosis technology from GenTegra^® LLC to preserve DNA extracts from one month to one accelerated year from different DNA quantities (from 1 ng to 0.2 ng) and sources (NIST, mocked samples, and true casework mixtures). We studied the quantity, integrity of DNA, and also the quality of the STR genetic profiles obtained. Results and Conclusions: Our results prove the high potential of this technology to preserve and to allow an effective recovery of the DNA extracts for forensic purposes.

1. Introduction

The use of DNA to help legal investigations has been a revolution since its first use in the United Kingdom in the Colin Pitchfork case [1,2], enabling a suspect to be found and a criminal to be identified. Today, the success of forensic genetics has led to considerable numbers of DNA samples to be analyzed and then to a considerable DNA storage issue. For example, the genetic casework unit of the forensic institute of the French gendarmerie (Institut de Recherche Criminelle de la Gendarmerie Nationale, IRCGN) analyzes more than 70,000 casework samples per year mainly from swabs that are fully consumed during DNA extraction. Since the end of 2023, it is a legal obligation in France to store the remaining DNA extract from a casework sample for up to 40 years [3] if a genetic profile does not match with a reference profile in the national database. This new regulation raises the question of the storage methods of these DNA extracts. The most common storage method to preserve DNA extracts for further analysis is freezing from −20 °C to −80 °C, especially for forensic purposes [4,5].

However, this storage technique has many associated risks and drawbacks. First, the necessary infrastructure must accommodate specialized freezers to maintain extremely low temperatures. Purchasing these freezers is a high initial investment along with regular maintenance costs and the need of spare freezers to prevent sample loss in case of failure, requiring staff availability. The monitoring system is also crucial to ensure that the temperature remains stable and adequate, with sophisticated alarms and sensors to detect any abnormal variations. In terms of energy efficiency, these freezers consume a lot of energy, which goes beyond the simple financial cost and raises environmental concerns. Sample transport is also challenging, as it requires cold transport systems to keep samples at constant, low temperatures. Any temperature fluctuations during transport can compromise the integrity of DNA, making this process difficult and expensive. Finally, freezing and thawing cycles can degrade DNA, making rigorous management essential to minimize these risks [6]. It is therefore useful to find an alternative storage system to negative temperature storage.

This preservation solution should ideally have a low cost of purchase or be equivalent to long-term storage in a freezer. It must also be easy to use at all stages of the process, i.e., from storage to the recovery of the DNA extract. It must enable the long-term preservation of very low amounts of DNA (less than 1 ng) at room temperature, while eliminating the constraints associated with cold chain storage. Finally, it must enable the full retrieval of the DNA extract, whether all at once or over multiple recovery times. No recent studies concerning the preservation of a low input of DNA (<1 ng) for forensic applications [7,8,9,10] have investigated DNA storage at room temperature. Frippiat et al. demonstrated that two products based on anhydrobiosis technology, QIAsafe (QIAGEN) and GenTegra^®-DNA (GenTegra^®), can be used to store DNA solutions for up to 6 months at room temperature, with good recovery yields and no obvious degradation. This study assessed the storage of DNA quantities as low as 3 ng [7]. However, with the increased sensitivity of current forensic genetic kits and protocols, it is now possible to obtain complete genetic profiles from quantities as low as 0.2 ng or even less [11]. Frippiat et al. later evaluated QIAsafe for the storage of very-low amounts of DNA (0.15 ng) up to 2 years at room temperature [10], but this product is no longer available. Lee et al. and Howlett et al. used SampleMatrix and DNAStable^TM products from Biomatricia^® Company, respectively, to store as little as 0.0625 ng and 1.4 ng of DNA. However, like QIAsafe, these products are no longer commercially available [8,9]. To date, no studies have evaluated the GenTegra^®-DNA product for its ability to store very-low amounts of DNA.

The GenTegra^® storage matrix is based on patented Active Chemical Protection™ (ACP™) formulations. According to the manufacturer, the proprietary matrix stabilizes biomolecules, particularly DNA and RNA, in a dry, ambient state by forming a protective coating around them. This coating provides a high level of protection from environmental factors. The range of DNA quantity that can be stored is from 0.05 µg to 25 µg.

Here, we assessed the GenTegra^® storage matrix to store DNA solutions at room temperature. The considered criteria were the ease of use, the DNA recovery yield, the integrity of DNA, and the quality of the STR genetic profile obtained. We treated several human DNA solutions or extracts from one month to one accelerated year from different DNA quantities (from 1 ng to 0.2 ng) and sources (NIST, mocked samples, and true casework mixtures).

2. Materials and Methods

2.1. DNA Sources

Two types of DNA sources were used in the experiments:

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DNA from the Standard Reference Material^® 2372a Human DNA Quantitation Component B, derived from a single female donor (National Institute of Standards and Technology (NIST)).

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A selection of DNA extracts from casework samples (previously analyzed and archived by the laboratory for judiciary purposes). For these samples, the French PJGN (Pôle Judiciaire de la Gendarmerie Nationale) ethics committee issued recommendations for these experiments and provided its approval on 11 September 2024 (recommendation n° 31).

2.2. DNA Extraction and Quantification

Casework samples were extracted using Crime Prep Adem-Kit (Ademtech, Pessac, France). The DNA from the NIST standard sample solution and the DNA extracts from casework samples were quantified by qPCR using the Investigator^® Quantiplex Pro Kit (QIAGEN, Hilden, Germany) both before and after storage. This kit also allows the evaluation of the DNA degradation rate. The initial degradation rate for casework samples used for experiments 2 and 3 are available, respectively, in Tables S1 and S2. Experiments were performed using half the volume of qPCR mix recommended by the manufacturer, i.e., 9 μL of qPCR mix plus 2 μL DNA. This is the in-house validated method for quantification at IRCGN.

2.3. PCR Amplification and Capillary Electrophoresis

The GlobalFiler^TM IQC kit (Thermofisher, Waltham, MA, USA) was used following the manufacturer’s recommendations. DNA amplification was performed with an Applied Biosystems™ Veriti™ Thermal Cycler (Thermofisher) for 30 cycles. Samples were prepared for capillary electrophoresis by adding 1 μL of the PCR product (or GlobalFiler™ Allelic Ladder) to 10 μL of formamide/size standard solution (Thermofisher). Prior to electrophoresis, the samples were denatured at 95 °C for 3 min then chilled on a cooling block for 3 min. Samples were analyzed on an Applied Biosystems™ 3500XL Genetic Analyzer (Thermofisher). Electrophoresis conditions were as follows: 1.2 kV, 24 s of injection, and migration at 13 kV for 1550 s. At the end of electrophoresis, the raw data obtained were analyzed using GeneMapper ID-X v1.6 software (Thermofisher).

2.4. DNA Storage and Recovery Protocol

The GenTegra^® matrix (GenTegra^® LLC, Pleasanton, CA, USA) was initially contained in a tube in a dehydrated form. Before the first use, 1.65 mL of ultra-pure water must be added. Once rehydrated, the solution should be stored at 4 °C and used within three months. The GenTegra^® storage matrix was used as follows: 15 µL of rehydrated matrix was put into a 200 µL well of a 96-well plate and dried for 24 h under a laminar flow hood at room temperature (average 20 °C, RT) and constant air humidity (average 35%) prior to sample deposit. Then, 30 µL of the sample solution was applied onto the matrix and dried for 24 h under a laminar flow hood at RT and constant air humidity prior to storage. Plates were sealed with a self-adhesive film and stored in the dark from 1 week up to 3 months at RT (20 °C; 35% humidity) or for 1 week to 69 days at 45 °C (45 °C; 12% humidity) (accelerated time corresponding to 1 month and 1 year, respectively). The Arrhenius equation was used to calculate accelerated aging [12,13,14,15].

DNA quantities used for experiments 1 and 2 were 1 ng, 0.5 ng, and 0.2 ng. For experiment 1, DNA from the NIST Standard B was in solution in ultra-pure DNA-free water. Each condition was tested in three independent experiments, with triplicates per experiment. For experiment 2, DNA from casework samples was in the elution buffer of the Crime Prep Adem-Kit. In order to observe the effect of time and temperature on the quantity of DNA stored, a casework sample was used to test all quantities for a specific period of time (for example, a sample was tested for a storage of one week at 1 ng, 0.5 ng, and 0.2 ng, either at RT or 45 °C). For the last experiment (experiment 3), the full volume of the remaining DNA extract solution from the casework sample was used, with volumes ranging from 20 µL to 38 µL depending on the available remaining volume of the sample after its first analysis for judiciary purposes. When the volume of DNA extract was more than 30 µL, the drying time was longer than 24 h.

Rehydration of the dried samples mixed with the matrix was performed with ultra-pure DNA-free water for 15 min at RT without agitation. The volume used was the same as the one used for loading: 30 µL for experiments 1 and 2, and from 20 µL to 38 µL for experiment 3.

2.5. Statistical Analysis

The results obtained are presented as the mean of several independent experiments. Several types of statistical analyses were used to compare the recovery yield for each quantity of DNA stored at each time. To check whether the distribution was normal, a Shapiro–Wilk normality test was performed on all experiments. Then, a t-test was performed to compare the recovery yield between DNA quantities recovered just after the dehydration time of 24 h (T0) and then between each storage time for a specific DNA quantity, and also between DNA quantities tested after a period of storage.

3. Results

3.1. Storage and DNA Recovery

3.1.1. Experiment 1: NIST DNA Solution

We first assessed the efficiency of DNA recovery after storage in the GenTegra^® matrix using DNA from the NIST Standard B in solution in ultra-pure DNA-free water. After the matrix deposition in each well, for each condition (1 ng, 0.5 ng, and 0.2 ng of DNA in 30 µL of ultra-pure DNA-free water), the samples were loaded and dried as indicated in Section 2.4. Each condition was tested in three independent experiments, with triplicates per experiment. All samples were quantified once, both before and after storage.

Figure 1 shows a high recovery of the DNA with more than 95% of the initial quantity loaded just after the dehydration time of 24 h (T0). No significant changes in DNA quantities were detected after the different storage times (p-value = 0.01) and no DNA degradation was observed. Recovery yield for each sample tested is available in Table S3.

3.1.2. Experiment 2: Calibrated Quantity of DNA from Casework Samples

Based on the results obtained, we then tested DNA extracts from casework samples at the selected quantities of 1 ng, 0.5 ng, or 0.2 ng. The same deposition and recovery protocol as NIST was used. Before and after storage, all the samples were quantified once. For each time and each quantity, the following numbers of samples were tested: 9 for T0, 29 for 1 week (stored either at RT or 45 °C), and 18 for 69 days (stored either at RT or 45 °C). Before and after storage, all the samples were quantified once.

Figure 2 shows again a high recovery of the DNA with more than 98% of the initial quantity deposited just after the dehydration time of 24 h (T0). After 1 week of storage, a significant decrease was observed compared to T0 for each quantity tested (p-value = 0.01) with more than 70% of the initial quantity recovered at least. However, the mean recovery yield remains stable compared to the 1 week RT results for each time and quantity tested, except for the 0.2 ng quantity stored for 69 days at RT (63% of recovery). Recovery yield for each sample tested is available in Table S4. No DNA degradation was observed (Table S1).

3.1.3. Experiment 3: Whole Remaining DNA Extracts from Casework Samples

Finally, for each DNA extracted from casework samples, we stored all the remaining solutions with random volumes and DNA quantities. The input volumes ranged from 20 µL to 38 µL and the DNA quantities ranged from 0.15 ng to 5.35 ng. After depositing the entire remaining extract volume, the samples were dried as indicated in Section 2.4. Hydration of the dried samples mixed with the matrix was performed with the same volume as the one used for loading, in ultra-pure DNA-free water for 15 min at RT without agitation. Before and after storage, all the samples were quantified once.

Regardless of the initial volume or DNA quantity deposited for storage, more than 85% of the initial quantity loaded was recovered after the dehydration time of 24 h (T0, n = 9) (Figure 3a). After 69 days of storage at RT or at 45 °C (corresponding to 1-year accelerated time), the recovery yields of DNA were more than 63% for an initial DNA input ranging from 0.15 ng to 0.49 ng, and more than 68% for higher quantities (n = 27 for each period of storage) (Figure 3b,c). We did not observe an effect of the initial volume used to load DNA on the GenTegra^® matrix. Again, no DNA degradation was observed (Table S2).

3.2. Quality of Genetic Profiles

For all DNA quantities and storage times studied with NIST DNA solutions, an exploitable STR genetic profile was obtained, matching the reference profile, with signal intensities proportional to the amount of the initial DNA amount used for PCR. Figure 4 shows the signal intensity of genetic profiles obtained after T0, 2 months at RT, and 69 days at 45 °C of storage. No artifacts were observed in the electrophoregrams. Genetic profiles were also analyzed at intermediate time points (1 month and 35 days at 45 °C) leading to similar results.

DNA profiles were also obtained from casework samples in experiments 2 and 3 at T0 and after storage for 69 days at RT or 45 °C. As the samples used came from unexploitable mixtures, it was not possible to compare the exact concordance of the profiles obtained between the original one and the “stored” one. Therefore, to enable a more robust and consistent assessment of DNA profiles before and after storage, comparisons were restricted to the profiles generated in experiment 3. These profiles exhibited highly similar overall patterns between the original analysis and after storage (Figure 5), indicating that the matrix does not exhibit any inhibitory effect following rehydration.

4. Discussion

The increasing use of DNA evidence, or more precisely the analysis of biological traces for human identification in forensic sciences, associated in France with a legal obligation to store DNA extracts up to 40 years [3] raises the question of the storage methods of these DNA extracts. For example, in France this represents more than thousands of samples that must be preserved each year. The storage by freezing at −20 °C or deep-freezing at −80 °C remains the standard and most commonly used method. Several studies [4,5] showed that extracted DNA stored at −20 °C for a long time enables its preservation in forensic samples. However, regarding the increasing number of samples to be stored, it is relevant to find an alternative to this storage method, which has a high economic and ecological cost over time, and is easier to use.

Different strategies exist to store DNA at room temperature; including lyophilization, inclusion in soluble matrices, like dextran sulfate; encapsulation in silica nanoparticles; and storage in an anhydrous and anoxic atmosphere in airtight capsules [16]. Some of these preservation strategies are expensive or difficult to implement because they do not allow for the mass preservation of DNA extracts.

This GenTegra^® solution uses a matrix that allows the dehydration of the DNA solution before long-term storage, simply by drying the sample in a biosafety hood. According to the manufacturer, the lowest amount of DNA that can be stored on the matrix is 50 ng. In 2011, Frippiat et al. evaluated the GenTegra^® matrix with 20 µL of a DNA solution loaded, but the lowest DNA quantity tested was 3 ng stored for 24 h (like our T0 condition) or 4.4 ng for 6 months at room temperature [7]. In our study, we demonstrate that GenTegra^® technology is able to store amounts of DNA as low as 0.2 ng up to 1 year in accelerated time. Because lower and lower amounts of DNA are often used in forensics to obtain a genetic profile using STR profiling (or NGS technology), we have also proven that samples stored using this technology lead to an exploitable STR genetic profile.

We first tested the storage of the NIST dilutions loaded on the GenTegra^® matrix to avoid any potential interaction of the extraction buffer with the matrix. In order to determine the impact of the buffer in which the DNA is in solution, we then studied the storage of DNA from casework samples extracted using the Crime Prep Adem-Kit. This kit is based on magnetic bead technology and is used routinely in our laboratory for extracting DNA from casework samples. Contrary to a previous study [7], we have not observed any negative impact of the extraction protocol using our magnetic bead system compared to DNA initially in solution in ultra-pure DNA-free water, regardless of the storage time.

Regarding long-term storage, McDevitt et al. showed that 3 µg of DNA can be effectively preserved at least 4 years at 25 °C [17]. However, it will be very useful to have results either in real time or in accelerated time, in order to quickly evaluate the benefit of using GenTegra^® technology for storing DNA forensic samples. This work is underway in our laboratory with storage studies of casework samples for 9 months either at room temperature or at 45 °C (4 years in accelerated time). It would also be interesting to test multiple cycles of dehydration/hydration after this long-term storage. Previous studies have proven that the DNA/GenTegra^® matrix mixture can be rehydrated at least three times (one cycle per 24 h) without affecting the recovery and the quality of the DNA without storage [7], but with higher quantities of DNA tested (1 µg).

In conclusion, our work demonstrates that the GenTegra^® matrix can preserve very-low quantities of DNA ranging from 1 ng to 0.2 ng at least for one year at room temperature, and that the extraction method using magnetic beads (and more particularly the elution buffer used) has no effect on the storage and recovery capacity. As the quantity needed for forensic genetic applications is decreasing, the storage and recovery of smaller quantities of DNA (0.1 ng and below) will need to be investigated. Several optimizations can also be considered, such as reducing the drying time or automating the protocol for high-throughput storage and recovery. Furthermore, these results indicate that samples can be used directly after rehydration and without removing the matrix from the sample. This kind of storage reduces the reliance on space, lowers freezer energy consumption, and facilitates the shipment of the DNA extract from one site to another. However, it remains to be determined whether this system enables long-term DNA storage over several years, which is currently under investigation in our laboratory.