Maximising DNA Extraction From Traces:

The Casework Direct System for DNA Methylation Level Estimation

Simona Severini, Federica Tommolini, Martina Onofri, Eugenia Carnevali

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INTRODUCTION

DNA methylation is becoming increasingly significant in the forensic field given the additional information, such as smoking habits or biological age, that can be inferred from its levels’ estimation at specific loci [1]. Different techniques are currently available for DNA methylation level quantification, the majority of which rely on bisulfite conversion, a chemical reaction in which sodium bisulfite deaminates cytosines into uracils [2–7]. The treated DNA can then be analysed through different methods, among which there is the Single Base Extension (SBE) technique [2,3,5,8]. Despite being an efficient, fairly straightforward, and easily implementable technique, bisulfite conversion-dependent SBE requires an amount of starting material (around 200 – 500ng of DNA), higher than the quantity that can be obtained from biological traces and in an that is sufficient for DNA profiling. However, a previous study [9] explored the sensitivity of the technique and the possibility of accurately analysing DNA methylation levels from a significantly lower amount of starting material. Ideally, DNA methylation levels quantification should be performed alongside traditional DNA typing, to optimize the information that can be inferred from a limited amount of available DNA.

The Casework Direct System (Promega) was developed to quickly extract casework evidence comprising of swabs and fabric cuttings. While its efficacy as an extraction method has been proven for touch DNA samples [10], our aim was to verify the kit’s efficiency in extracting from casework samples a quantity of DNA suitable for methylation levels analysis through bisulfite conversion-dependent SBE. Additionally, alternatives to the protocol were considered to obtain a DNA extract that is improved, in its quality and quantity, for the downstream analysis. In this study, DNA methylation level estimation was performed for age prediction purposes, given the relevance that biological age estimation has gained in the forensic field. In particular, five CpG sites (ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59) were analysed, based on both a protocol and tool already the subject of extensive work in our laboratory [8].

MATERIALS AND METHODS

Two types of casework samples were set up: dried blood spots and smoked cigarette butts. A volunteer of 67 years of age donated a vial of peripheral blood (vacutainer purple topped, 3.5mL). 1mL of blood was spotted on a sterile cotton towel and allowed to air dry, then placed in an evidence paper bag and stored in a dry, dark place at room temperature for 2 months. The same volunteer was asked to smoke four cigarettes on different days and to put the cigarette out on an ashtray, previously sterilised with bleach and exposed to UV lights for 30 min. The cigarette butts were then collected, placed each in a separate evidence paper bag, and stored in a dry, dark place at room temperature for 2 months.

The samples consisted of four 1cm2 towel cuttings for blood spots and the paper sheaths around the filter, for the four cigarette butts. The samples were extracted using the Casework Direct System (Promega) according to the manufacturer’s instructions. Both kinds of samples were cut into smaller pieces for the samples to be completely covered by the Casework Direct Solution and thus to aid the extraction process. Considering the well-known inhibition that haematin and other debris have on downstream reactions, the kit’s suitability for DNA methylation analysis was evaluated in three steps. First, in Phase I, the DNA extracts obtained as per the manufacturer’s instructions were analysed. Subsequently, modifications were performed not to the kit’s protocol directly: either the substrate was pre-treated before the extraction (Phase II) or the DNA lysates were further processed afterwards (Phase III), discussed as below and graphically represented in Figure 1. These modifications were performed keeping in mind that, the type of substrate, debris, carry-over, and haematin, could have an adverse effect on the bisulfite conversion reaction, the most crucial step in the DNA methylation analysis. Therefore, the pre-treatment of Phase II and downstream treatment of Phase III were carried out with the aim of purifying as much as possible the lysate and thus obtaining optimal results in the quantitative analysis of DNA methylation levels.

PHASE I) Extraction as per manufacturers’ instructions:

One blood cutting and one cigarette filter paper were extracted using 400μL of Casework Direct Solution per the kit’s protocol. To get a tentative idea of the suitability of the lysate for DNA methylation levels estimation, the obtained lysates were quantified and underwent downstream analysis. Based on the results, two different modifications were explored in Phase II and III.

PHASE II) Pre-treatment of samples with water:

One blood cutting and one cigarette filter paper were placed in a 1.5mL tube, then filled with sterile water up to volume. The samples were placed in a thermal shaker at 56°C for 3 hours. This pre-treatment step aimed at washing the DNA off from the substrate so that blood cells and debris (from the filter paper, for example) could be separated prior to the extraction. Therefore, the samples were placed in a DNA IQ™ Spin Basket (Promega) and centrifuged at 14, 000rpm for 10 minutes. The Spin Basket with the substrate was eliminated and the supernatant was discarded. The obtained pellet was resuspended by using 200μL of the Casework Direct Solution and incubated, with agitation, at 70°C for 30 minutes. The samples underwent quantitation and DNA methylation estimation downstream analysis.

PHASE III) Purification and concentration of the CDS kit’s lysate:

Two blood spots and two cigarette filter papers were extracted with 400μL of Casework Direct Solution as per the kit’s protocol. The lysates obtained by standard Casework Direct System were processed with the aim of obtaining adequately purified DNA lysates, suitable for bisulfite conversion. Two different purification approaches were employed:

  • A) Amicon® Ultra-0.5 Centrifugal Filter 100kDa MWCO: One blood spot and one cigarette paper lysates were diluted with sterile water to the volume of 500μL and then purified by using the Amicon® Ultra-0.5 Centrifugal Filter 100kDa MWCO (Millipore) according to the product’s protocol. Around 30μL of purified and concentrated lysate was obtained for each sample.
  • B) DNA IQ™ System: One blood spot and one cigarette paper lysates were aliquoted in 1.5mL tubes so that, based on quantitation data from Phase I, each aliquot would contain around 100ng of DNA. In order to obtain the DNA amount necessary for bisulfite conversion, two aliquots per type of sample were processed with the DNA IQ™ System (Promega) as follows. Prepared Lysis Buffer and the DNA IQ™ resin were added to the aliquoted lysates in the recommended quantities of the DNA IQ™ System protocol. Samples were incubated, with agitation, at 37°C for 15 minutes and then placed on the magnetic stand. Washes and elution steps were executed as per the product’s protocol. Each aliquot was eluted with 30μL of elution buffer and, subsequently, the eluted DNA from both aliquots of each sample were reunited for a total of 60μL of purified DNA for both the blood spot sample and the cigarette filter sample.

Figure 1: Graphical representation of the extraction trials carried out with the Casework Direct System (CDS). Phase I: extraction performed with the Casework Direct System only; Phase II: samples were soaked in sterile water at 56°C for 3 hours, then centrifuged at 14,000rpm for 10 minutes. The pellet was resuspended with 200μL of Casework Direct Solution and incubated at 70°C for 30 minutes, as per kit’s protocol; Phase III: The samples were extracted according to the CDS kit instructions and the purified by using A) Amicon® Ultra-0.5 Centrifugal Filter 100kDa MWCO (Millipore) and B) DNA IQ™ System (Promega).

DNA extracts from all Phases were quantified by using the PowerQuant® System (Promega) and quantitation results are reported in Table 1. The samples were then bisulfite converted by using the EZ DNA Methylation-Direct kit (Zymo Research): given the amount of DNA obtained, lower than the threshold suggested by the kit’s manufacturer, the samples were generally converted undiluted. An exception were the the blood spot samples extracted in Phase I and Phase III-A, diluted 1:2 and 1:4 with sterile water, respectively, given the appearance of the lysates to dilute possible inhibitors of the bisulfite conversion reaction. DNA levels were estimated for five loci (ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59) by using the Single Base Extension technique according to the protocol by Onofri et al. [8]. Fragments were then separated by using the SeqStudio™ Genetic Analyzer (ThermoFisher Scientific).

After establishing that the best results in terms of quality and quantity of yielded DNA consisted of the CDS lysate purified with the DNA IQ™ System, an additional conversion for each sample was carried out. Additionally, two amplifications per converted DNA sample were performed, for a total of four replicates for each blood spot and the cigarette filter paper samples. The samples were analysed in replicates as it was suggested by [8] for a more accurate methylation level estimation and subsequent age prediction. The DNA methylation level obtained were then inserted in the tool developed by Onofri et al. [8] in order to check for age prediction accuracy. Results are reported in Table 2.

Table 1: Samples were quantified by using the PowerQuant® System (Promega). Here quantitation data, amount of converted and amplified DNA (ng) are reported.

Table 2: Methylated DNA ratio calculated per each locus and per each replicate. Both samples were converted an additional time, for a total of two converted eluates per DNA sample, and amplified twice per converted DNA, as suggested in [8]. C1 and C2 indicate the converted DNA replica, while R1 and R2 refer to the amplified replica. The predicted age was calculated by using the tool by Onofri et al. [8] and the prediction error was calculated as a Mean Absolute Deviation (MAD). The MAD was calculated as the mean of absolute values of the difference observed between the predicted age and chronological age across all four replicates per samples.

Quantitation data obtained from all phases is reported in Table 1. As can be observed, the Casework Direct System used as per the manufacturer’s instructions yielded the second-highest amount of DNA for both types of samples. However, given the nature of the extraction method, some debris was carried over in both samples. The blood spot extracted with the Casework Direct System presented as a dark red lysate, with a viscous substance deposited at the bottom of the tube. Only the clear solution was pipetted, and it was additionally diluted 1:2 with sterilised water before being converted. After the dilution, the presence of haematin, red blood cells and other debris, however small, may have caused an inefficient conversion and downstream analysis. As per Figure 2, only two complete loci with very low intensity peaks and one incomplete locus were observed for the blood spot extract. The cigarette filter paper lysate appeared to be clear, however, some debris was present as well, realistically from the partial breaking of the paper. In this case as well, only the supernatant was converted and, as it can be observed in Figure 3, the electropherogram presented peaks for all five loci, even though non-specific peaks and high background noise were present. The data obtained were inserted in the tool by Onofri et al. [8] and it returned a predicted age of 68.5 years (not reported in Table 2), within the expected prediction error indicated in [8].

As it can be expected, the samples concentrated and purified by using the Amicon® Ultra-0.5 100kDa MWCO (Millipore) yielded the highest results. Despite the partial purification, the DNA lysate obtained for both the blood spot and the cigarette paper, still presented a debris at the bottom of the tube. In both cases, only the supernatant was pipetted to be converted and, additionally, the blood spot lysate was diluted 1:4 prior to bisulfite conversion. As it can be observed from Figures 2 and 3, the EPGs did not present peaks in all five loci: two complete loci with very low intensity peaks and one incomplete locus for the blood spot samples, and only one peak for the filter paper. It is hypothesised that the presence of debris, despite being minimal and not flagged by the PowerQuant® System, may cause problems during the conversion step. Particularly, in the case of the cigarette paper, the residues were more diluted in the Casework Direct System lysate than in the Amicon® 100kDa eluate. This consideration is particularly relevant considering that the Casework Direct System lysate electropherogram presented peaks in all five loci despite half the quantity of DNA being converted in comparison to the Amicon® 100kDa eluate and given that the amount of template DNA in the SBE reaction was the same in both cases.

Figure 2: Electropherograms of the blood spot lysates extracted according to the three phases of the study. CDS is an abbreviation for “Casework Direct System”.

Figure 3: Electropherograms of the cigarette filter paper lysates extracted according to the three phases of the study. CDS is an abbreviation for “Casework Direct System”.

The samples pre-treated with a water bath and then extracted with the Casework Direct System reported the lowest DNA concentrations. The blood spot lysate presented a flat electropherogram while the cigarette paper lysate yielded peaks in three out of five loci, which is understandable given the scarce DNA amount converted and amplified. Lastly, the combination of both the Casework Direct System and the DNA IQ™ System yielded the best results in terms of electropherograms completeness for both samples. As expected, a loss of DNA was observed: for both samples, 200ng of DNA (albeit in different starting volumes) were purified and only 54.6ng and 108.6ng of DNA for the blood spot and the cigarette paper, respectively, were recovered. These results support the assumption that carry-over and debris could cause issues during the bisulfite conversion step. In fact, despite the very low DNA amount converted and amplified, the blood spot cutting presented peaks at five out of five loci even if non-specific peaks were present and the cigarette paper sample resulted to be much cleaner than the other electropherogram obtained with the Casework Direct System-only extraction method.

As mentioned in the previous section, the blood spot cutting and the cigarette paper lysates purified with the DNA IQ™ System were converted once more for a total of two converted replicates for type of substrate and each converted DNA was amplified twice, for a total of four replicates per substrate. All replicates for both samples predicted age correctly with a mean absolute deviation (MAD) of 0,733 years for the blood spot’s replicates and 0,323 for the cigarette paper’s replicates.

CONCLUSIONS

The exploratory nature of this study proved how the Casework Direct System (Promega) can be implemented as a fast extraction alternative in the DNA methylation levels estimation workflow. In particular, this kit seems to be efficient in providing an amount of DNA suitable for methylated DNA analysis, especially in casework evidence presenting buccal cells and saliva on them, such as smoked cigarette filters. Other types of substrates, like fabric cutting soaked with dried blood, can be, nonetheless, easily extracted yielding DNA lysates of suitable quality and quantity, with a minor modification to the kit’s protocol. In fact, the addition to the protocol of a rapid purification step with the DNA IQ™ System allowed to obtain much cleaner electropherograms and peaks in all the loci analysed, despite the expected DNA loss in the process.

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Simona Severini Biology Specialist in Clinical Pathology and Clinical Biochemistry

Since 2007 I have been attending the Forensic Genetics Laboratory of the S. Maria hospital in Terni which in 2020 obtained the UNI CEI EN/ISO IEC 17025 Accreditation. After obtaining a Master’s Degree in Biology and PhD in Forensic Genetics, I continued my training in this field, regularly participating in congresses, training courses, proficiency tests, and research projects as well as the drafting of various scientific publications. In particular, my topics of interest include kinship statistics, mixture interpretation, and biogeographical ancestry analyses. In recent years, alongside routine casework activities, my research projects focused on the study of touch DNA with particular interest on the activity level topic and on DNA methylation evaluation for the age estimation of a trace’s donor.