DNA Purification Cell Lysis and Wash Step Modifications for Low-Template DNA Sample Processing

Abstract

As DNA technology becomes increasingly sensitive, forensic laboratories are receiving more low-template DNA samples. These samples, already low in DNA content, become even more challenging to process as the available DNA becomes further reduced during the extraction step. In this study, two extraction modifications were tested to determine if the cause of DNA loss could be identified and mitigated. A double lysis technique was used to test for DNA loss in the sample collection substrate, and lysate eluates were re-extracted to determine DNA loss from inefficient binding to the silica column. Both modifications showed DNA was lost at these steps. However, resulting STR profiles from these samples had fewer peaks and lower peak heights when compared to samples processed with no extraction modifications. Overall, the potential benefits of adding these extraction modifications for low-template DNA sample processing are not enough to justify the risk associated with additional manipulation.

As DNA detection methods become increasingly sensitive, forensic laboratories are receiving larger numbers of low-template DNA samples that, until recently, would have little hope of producing any short tandem repeat (STR) profile results. Unfortunately, many low-template DNA samples are still more likely to result in incomplete or even null STR profiles. In addition to the limited amount of starting DNA, current sample collection and extraction methods further reduce the amount of DNA available for PCR amplification. Manufacturers of common forensic DNA extraction kits, such as QIAGEN, have reported unavoidable loss of approximately 20% of input DNA (1). However, other independent groups have reported DNA loss closer to 70% after extracting known quantities of DNA using these kits (2,3). Currently, many of the studies regarding DNA extraction efficiency focus on comparing the methods themselves rather than where any potential DNA loss is actually occurring (2,4-6). Furthermore, most of the available literature focuses on DNA yields from ancient bones or soil, which have other factors affecting extraction efficiency compared to more common forensic substrates (4-7).

Some DNA recovery studies have focused on the substrate (used for sample collection) as the primary culprit for DNA loss. It has been suggested that DNA is being trapped in the substrate itself and that the swab material, such as tightly wound cotton fibers, can affect the ability to efficiently retrieve DNA useable for downstream STR analysis (8-12). One study reported that over 50% of the usable DNA may not be released from the swab substrate during the lysis step (12).

Alternatively, DNA loss could also occur in the silica column itself when performing solid-phase extraction methods; however, there is little information on which step of the extraction process is most responsible, or if it is caused by excessive tube transfers and manipulation (13). It is possible that not all the DNA is properly binding to the column when the sample lysate is filtered through, or that some DNA is prematurely released from the column during subsequent washes. Lastly, a portion of the DNA may not be released from the column during the final elution step. One study comparing extraction kits reported that the DNA yields increased as the amount of silica in the column increased implying that inefficient initial binding of DNA to the silica (or possibly an overloading of the silica column in the case of high-yield DNA samples) could be a primary source of DNA loss (2).

The aim of this study was to evaluate potential sources of DNA loss during the extraction process and to determine if this loss was recoverable for low-template samples. Specifically, modifications to the cell recovery/lysis step and the silica column wash steps were examined.

Methods

Sample Collection

Fingerprints from volunteers were collected on glossy paper. Half of each set of fingerprints from each individual were left untreated while the other half were visualized with magnetic powder (EVIDENT, Union Hall, VA). For the visualized samples, magnetic powder was dusted onto the fingerprints using a magnetic applicator (EVIDENT). Finally, for all samples, fingerprint tape (EVIDENT) was placed directly onto the paper and samples were stored as archived latent fingerprints (ALFPs). Fingerprints were stored undisturbed in a separate enclosure at room temperature for two years prior to DNA processing. A reference buccal swab was also collected from each volunteer fingerprint donor; buccal swabs were subsequently used to develop reference STR profiles for comparison in each of the studies detailed below.

Optimized DNA Extraction and Purification

One untreated ALFP sample and one magnetic powder-treated AFLP sample from each individual donor were processed for DNA analysis using a workflow previously optimized for ALFP samples (14). Briefly, for the optimized method, the paper and tape were disassembled and cut into small squares (approximately 3 mm × 3 mm in size). The tape and paper cuttings were then placed in separate tubes and lysed overnight at 56°C using 300 μL Buffer ATL (QIAGEN, Hilden, Germany) and 20 μL Proteinase K (QIAGEN). The tape and paper were removed from the lysate, placed in a spin basket, and centrifuged at 6000 × g for 1 min to remove any remaining liquid. The lysate then was placed on a column, and DNA was extracted using the QIAamp® DNA Investigator Kit (QIAGEN) with a final elution volume of 25 μL. After quantification (described below), all ALFP samples were further purified using Centri-Sep columns (Prince Separations, Adelphia, NJ). For this, the columns were rehydrated with 800 μL sterile ddH₂O and incubated at room temperature for 2.5 h. The bottom of each column was then opened and excess water allowed to flow out. Columns were then spun at 800 × g for 2 min. Last, DNA samples were loaded onto separate columns and they were again spun for 2 min at 800 × g. After purification, the samples were concentrated, as needed, using a Savant™ DNA 120 SpeedVac™ (Thermo Fisher Scientific, Waltham, MA) vacuum centrifuge to obtain a final concentration of 0.2 ng/μL or if DNA was not detected, samples were concentrated to a final total volume of 5–8 μL.

DNA Quantitation, Amplification, and Analysis

All DNA samples were quantified using the Investigator® Quantiplex Kit (QIAGEN) on an ABI PRISM® 7500 thermocycler using SDS v1.2.3 software (Thermo Fisher Scientific) following the manufacturer’s protocol (15), but using a half volume reaction. DNA yields were calculated by multiplying the concentration by the elution volume, and a mean yield was calculated for each experimental group. STR amplification was achieved using the AmpFlSTR® Identifiler® Plus PCR Amplification Kit (Applied Biosystems, Foster City, CA) on a GeneAmp® 9600 PCR System (PerkinElmer, Waltham, MA) following the manufacturer’s protocol, (16) but with half volume reactions and a 45 min 60°C final extension. Target DNA input for all amplifications was 1.0 ng (including all buccal swab samples processed). Individual fingerprint samples with total DNA yields <1.0 ng were concentrated as described above, and the entire sample was used for amplification.

STR amplicons were separated on an ABI PRISM® 3130 Genetic Analyzer (Applied Biosystems) and analyzed with GeneMapper® ID software v4.1 (Life Technologies, Carlsbad, CA). For capillary electrophoresis, 3 μL amplified DNA was combined with 0.1 μL GeneScan™ 500 LIZ® Size Standard (Life Technologies) and 10.5 μL Hi-Di formamide (Life Technologies). Samples were injected for 15 sec at 3 kV and electrophoresed 25 min at 15.0 kV on a 36 cm capillary filled with POP-4™ polymer (Applied Biosystems). All samples were analyzed using previously validated thresholds (50 RFU analytical threshold and 250 RFU stochastic threshold). Data were compared to reference samples to determine the percent of expected STR peaks detected (Amelogenin was not included in data analysis). An average STR allele peak height was calculated for each sample (homozygous peak heights were halved). Known, well-characterized artifacts were excluded from calculations. Quantitation and STR data for the modified protocols were compared to samples processed using the optimized workflow (14) with no modifications, which served as a control to determine if improvements to the obtained profiles were made. Differences between experimental groups were compared using a Student’s t-test (α = 0.05).

Evaluation of DNA Loss

Double Lysis

One magnetic powder-treated and one untreated aged ALFP (from glossy paper) taken from ten volunteers were processed for DNA analysis following the optimized workflow described previously (14) and above, but with several modifications (referred to herein as “double lysis”). For these samples, after the initial overnight lysis, the lysate was removed and stored at 4°C, and the cuttings were lysed a second time under the same conditions. After the second lysis, the two lysates were combined and purified over a single QIAamp® DNA Investigator column (QIAGEN) and processed as described above. Quantitation and STR data obtained from this experiment was compared to that from the unmodified optimized workflow to determine if any improvements were observed.

Combined Wash

Initially, a preliminary study was conducted using buccal swabs collected from ten individuals. DNA from these samples was extracted using the optimized ALFP method described previously (14) and above but with several modifications. During the extraction process, each wash (lysate eluate, AW1, AW2, and ethanol) was retained and was extracted as a separate sample on a separate column, as described above. After quantification, the sum of DNA yields from each wash sample was added to the original extract yield to obtain the total DNA yield. The percentage of total DNA captured from each individual wash step was calculated to identify where, if at all, measureable DNA was lost during the wash process.

Based on the information obtained in the preliminary study, a second study commenced using ALFP samples. For this study, one magnetic powder-treated ALFP and one untreated ALFP from each of ten volunteers were processed following the optimized workflow; however, the original lysate eluate from each sample was retained and processed as a separate DNA sample. The original extract and the lysate extract from each sample were initially quantified separately as described above; following quantification, the two DNA extracts were combined into a single sample and were then quantified again (as described above). The combined sample was then processed following the optimized workflow described previously (14) and above.

Results and Discussion

Double Lysis Modification

In an effort to improve the release of biological material from the paper and adhesive substrates, aged ALFP lift card samples were processed using a double lysis DNA extraction protocol. When compared to the previously described optimized workflow for ALFP samples (14), which includes only a single lysis step, the double lysis method showed a ~26% increase in the number of samples with quantifiable DNA (Table 1). However, of those samples with quantifiable DNA, the double lysis method produced lower DNA yields (Table 1, p ≥ 0.15 for both treatment groups). While this difference is not statistically significant, there is a potential practical impact given that samples processed with the double lysis method were more likely to result in total DNA yields that were substantially below the recommended DNA input for STR multiplex amplification (16). When STR profiles were examined, samples processed using the double lysis method produced nearly half the number of expected STR alleles than those processed using a single lysis (optimized method) (Table 2, p = 0.04). Additionally, those samples processed using the double lysis method produced STR allele peaks that were significantly lower (189 ± 183 rfu) versus those obtained from samples processed using a single lysis step (761 ± 823 rfu), bringing the peak heights much closer to the analytical threshold and below stochastic threshold in many cases (Table 2, p = 2.2e⁻²³). It is possible that the second lysis step facilitated the release of additional DNA allowing for detection of low levels of DNA from more samples, which in turn, caused a concomitant decrease in the average yields and allele peak heights from samples that may have otherwise shown no detectable DNA. These data show that, while an additional lysis step may improve the ability of the assay to dislodge biological material from the initial substrate, the additional step does not result in an improved ability to obtain a quality STR profile.

TABLE 1—

Effect of double lysis on DNA yields.

Extraction Method	Samples with DNA Detected (%)	Average DNA Yield (ng)
		Magnetic*	Untreated†
Optimized (n = 35)	69.44	0.918 ± 1.44	0.481 ± 0.715
Double lysis (n = 20)	95	0.270 ± 0.266	0.328 ± 0.366

^*p = 0.15.

^†p = 0.53.

TABLE 2—

Effect of double lysis on STR success.

Extraction Method	Samples with Detected Alleles (%)	Expected STR Alleles Observed (%)*	Average Peak Height†
Optimized (n = 35)	55.56	51.29 ± 38.21	760.61 ± 822.98
Double lysis (n = 20)	50	28.37 ± 22.24	188.88 ± 183.39

^*p = 0.04.

^†p = 2.2e⁻²³.

Combined Wash Modification

In order to identify potential areas of DNA loss during the extraction process, an initial study was conducted using high-yield buccal swab samples. Data from this study revealed that 95.7% of the total available DNA from the buccal swabs was retained in the original sample extract (Table 3). However, an additional 4.3% of the total accessible DNA was obtained from the initial lysate eluate, which is typically discarded. In the case of high DNA yields, 4.3% is easily enough DNA to independently produce a complete STR profile. Unfortunately, subsequent washes produced negligible amounts of DNA, with a combined total of <0.01%. These data indicate that the most substantial DNA sample loss that occurs during the purification using this column-based method results from a failure of all available DNA to initially bind to the silica when the lysate is first added and spun through the column. Further, while a small amount of DNA is detached during subsequent washes of the column, it is not enough to meaningfully contribute to the low yields that are often obtained from more challenged or low-template forensic samples.

TABLE 3—

Percentage of total DNA recovered from each step of QIAamp® DNA Investigator Kit extraction.

Wash (n = 10)	Percentage of total DNA
Original extract	95.73 ± 4.54
Lysate	4.26 ± 4.54
AW1	0.004 ± 5.8e−5
AW2	0.0004 ± 3.3e−6
EtOH	0.001 ± 5.6e−6

Based on the data from the preliminary study, a study using aged ALFP lift card samples was conducted. In this study, the initial lysate flow-through was retained and processed as a separate DNA sample; after quantification, this lysate flow-through DNA was combined with the original DNA extract from the same sample. As shown on Table 4, when the lysate eluate DNA sample was combined with the original extract, there was a decrease in the overall DNA yields (p = 0.07) even though more samples produced detectable DNA. Further, ALFP lift card samples processed with the combined DNA extract produced STR profiles that yielded significantly fewer expected STR allele peaks and a significantly reduced mean allele peak height (Table 4, p = 0.02 and p = 1.1e⁻²⁶). It is likely that the added manipulation and the increased volumes that result from combining the two extracts led to this net negative effect. Ultimately, this data shows that rescuing the lost DNA and combining it with the original sample extract results in less informative STR profiles than those obtained from control groups processed without this modification.

TABLE 4—

Effect of combining sample extract with lysate eluate on DNA yield and STR profile.

Extraction method	Samples with DNA Detected (%)	Average DNA Yields (ng) ±SD*	Samples with Detected Alleles (%)	Expected STR Alleles Observed (%)†	Average Peak Height‡
Optimized (n = 36)	69.44	0.700 ± 1.132	55.56	51.29 ± 38.21	760.61 ± 822.98
Combined extract (n = 20)	95.00	0.263 ± 0.228	50.00	23.27 ± 15.66	140.03 ± 101.93

^*p = 0.07.

^†p = 0.02.

^‡p = 1.1e^−26.

Conclusion

In these studies, minor extraction procedure modifications were employed to attempt to identify areas of DNA loss that could be easily remediated in an effort to further improve DNA yields and downstream STR profiles. While our studies confirm that cellular material/DNA remains trapped within the substrate after initial cell lysis and remains in the lysate flow-through after initial centrifugation through a silica column, the modifications tested herein did not prove beneficial enough to justify the additional time needed or the increased risk of contamination that comes with additional sample manipulation.