Elimination of contaminating amplified short tandem repeat products by autoclaving and ultraviolet irradiation

Abstract

DNA contamination can result in false interpretation of short tandem repeat (STR) DNA typing. Proper decontamination is particularly required in forensic DNA laboratories where probative value of the evidence may be affected. The aim of this study was to establish an effective DNA decontamination procedure for amplified STR products focusing on laboratory-related contamination. We verified the effectiveness of thermally and temporally extended autoclaving and ultraviolet irradiation for the elimination of contaminating amplified STR products. STR amplification products were prepared using a control genomic DNA template and generated using the AmpFℓSTR^® Identifiler^® Plus and Yfiler^® polymerase chain reaction amplification kits. In this study, the contaminants were dried before decontamination treatment, which resembles actual contamination situations. One microlitre of amplified STR products was eliminated by a combination of autoclaving (128°C, 420 min) and UV irradiation (60 J/cm²). Our results reveal that the combination treatment represents an effective DNA decontamination procedure and a practicable method in standard level laboratories. Finally, we propose a comprehensive approach for forensic DNA laboratories to implement to minimise contamination issues and guarantee provision of authentic results.

Keywords

Forensic DNA typing contamination STR amplified products autoclaving UV irradiation

Introduction

DNA contamination is a crucial issue in forensic casework. DNA typing, wherein short tandem repeat (STR) loci are amplified by multiplex polymerase chain reaction (PCR), is used for human identification.¹ High sensitivity PCR-based DNA typing is suitable for trace DNA samples. However, it is also susceptible to DNA contamination.¹ Contamination of case-related and/or -unrelated DNA can result in false interpretation, such as misidentification and mixed DNA profiles. In addition, foreign DNA derived from manufacturers, investigators and case samples may contaminate irreplaceable forensic evidence via laboratory instruments and consumables during sample collection and processing. Therefore, it is essential for forensic laboratories to take effective and stringent measures to prevent false interpretation and losing probative value of the evidence due to DNA contamination.

To avoid DNA contamination-associated errors, forensic laboratories employ systematic approaches to prevent, identify and avoid error recurrence. Typical strategies for preventing contamination involve cleaning laboratory surfaces, using certified DNA-free consumables and physically separating work processes and areas.^1,2 While these anti-contamination measures are effective, DNA contamination cannot be completely prevented. Thus, laboratories need to detect DNA contamination by monitoring DNA extraction and PCR amplification controls, cross-checking DNA profiles between cases/samples and comparing DNA profiles against elimination databases.^1–3 Also, case examination for the incidence of DNA contamination and quality control of facilities and laboratory staff are important to maintain the risk-management system for avoiding further errors. Eventually, elimination of residual DNA prior to use of laboratory instruments and consumables remains the most reliable approach to prevent DNA contamination.^4–7

Eliminating preventatively PCR-amplifiable DNA from laboratory instruments and consumables requires effective methods in forensic DNA typing. Several studies have evaluated the effectiveness of sterilisation techniques for DNA decontamination, including ultraviolet (UV), gamma and electron-beam irradiation, ethylene oxide treatment and autoclaving.^4,5,8–13 Autoclaving and UV irradiation are easy-to-use methods, widely applied for eliminating DNA prior to use of instruments and consumables in forensic laboratories. Gefrides et al.⁴ reported that autoclaving for 120 min at 121°C eliminated PCR-amplifiable DNA from 10 µL of dried saliva. However, human bodily fluids are not the sole source of contaminating DNA in forensic laboratories. Other sources include DNA extracts and amplified STR products. In particular, amplified STR products are presumably difficult to eliminate due to their extremely high copy number. Moreover, PCR amplicon sizes of STR markers are designed to be as small as possible in commercial DNA typing kits for forensic purposes.⁹ Currently, there is no appropriate strategy established for the elimination of amplified STR products. Once established, laboratories will be able to adopt appropriate approaches to prevent amplified STR product-induced contamination.

The objective of this study was to define a DNA decontamination procedure effective against STR amplification products. We investigated the effectiveness of thermal (121–128°C) and temporal (20–420 min) parameters of autoclaving and UV irradiation energy (10–60 J/cm²) to eliminate PCR-amplifiable DNA derived from amplified STR products.

Methods

Sample preparation

Amplified STR products were generated using 0.5 ng of DNA 9947A (positive control), 0.5 ng of DNA 007 (positive control) or 10 µL of ultrapure water (negative control). Amplification was performed using a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA) with the AmpFℓSTR^® Identifiler^® Plus PCR amplification kit and the AmpFℓSTR^® Yfiler^® PCR amplification kit (Applied Biosystems) following the manufacturer’s protocols. Ten microlitres of the amplified STR products derived from control DNA 9947A was serially diluted with ultrapure water from neat to 10⁶-fold dilution (corresponding to 10–0.00001 µL of the amplified STR products). Dried amplified STR products were prepared from 10 µL of the amplified STR products which were adhered to 0.2 mL polypropylene PCR tubes, latex gloves and cotton swabs and heated at 95°C for 1 h using a GeneAmp PCR System 9700 thermal cycler. Additionally, 1 µL of allelic ladders from the AmpFℓSTR^® Identifiler^® Plus PCR amplification kit and the AmpFℓSTR^® Yfiler^® PCR amplification kit were similarly dried.

Autoclaving and UV irradiation

An SM-32 (Yamato Scientific, Tokyo, Japan) autoclave was used in this study. Dried amplified STR products were treated for 20, 300 or 420 min of autoclaving at 121°C and 128°C as follows. Dried amplified STR products in uncapped 0.2 mL tubes were placed in the autoclave standing on a tube rack, sealed with a sterilisation bag and autoclaved. Subsequently, autoclaved samples were dried again by heating at 95°C for 1 h using a GeneAmp PCR System 9700.

Dried amplified STR products were exposed to 10, 20, 30, 40, 50 or 60 J/cm² of UV irradiation using a DNA-FIX DF-254 (Atto, Tokyo, Japan) transilluminator as follows. UV doses of >10 J/cm² required sequential UV irradiation because 10 J/cm² represents the maximum energy the instrument can deliver. Dried amplified STR products in uncapped 0.2 mL tubes were placed in the transilluminator standing on a tube rack and UV irradiated. Distance from the UV light source was approximately 10 cm.

To verify the effectiveness of the combination of autoclaving and UV irradiation, 1 µL of the dried amplified STR products (attached to PCR tubes, latex gloves and cotton swabs) was treated using the following procedures. After autoclaving (128°C, 420 min), the samples were dried again and exposed to 10–60 J/cm² of UV irradiation, as above.

Existing DNA decontamination techniques

To compare the effectiveness of DNA decontamination procedures for amplified STR product elimination, we applied other existing anti-contamination techniques to the dried amplified STR products (attached to PCR tubes, latex gloves and cotton swabs), that is, treatments with bleach (6% NaHClO), ethanol or DNAZap™ solutions (Life Technologies, Carlsbad, CA). Each 1 µL of the dried amplified STR products in tubes was soaked in 100 µL of these solutions and incubated for 5 min at room temperature. After discarding these decontamination solutions, the treated samples were washed with 200 µL of ultrapure water and dried.

STR typing

Autoclaved, UV-irradiated and bleach-, ethanol- or DNAZap™-treated and untreated samples were dissolved in 10 µL of ultrapure water and amplified using the AmpFℓSTR^® Identifiler^® Plus PCR amplification kit or the AmpFℓSTR^® Yfiler^® PCR amplification kit according to the manufacturer’s specifications. The amplified STR products were separated by capillary electrophoresis using a 3130xl Genetic Analyzer and the data-collection software 3.0 (Applied Biosystems). Injection time and voltage were 10 s and 3.0 kV, respectively. Run data were analysed using GeneMapper ID software 3.2.1 (Applied Biosystems), with a peak detection threshold of 150 relative fluorescent units.

To deepen the reason of artefacts from autoclaved samples, no amplification controls were prepared. Autoclaved samples were dissolved in 10 µL of ultrapure water and mixed with 15 µL of the amplification master mix. Immediately after mixing, 1 µL of the mixture (no amplification controls) was separated by capillary electrophoresis, as described above, without PCR amplification.

Results

Autoclaving

We tested the effectiveness of thermally and temporally modified autoclaving for DNA elimination of amplified STR products for forensic purposes. Under standard conditions of autoclaving used for microbial sterilisation (121°C, 20 min), PCR-amplifiable DNA of dried amplified STR products (corresponding to 10–0.001 µL) was not eliminated. Amplicons of D8S1179, D3S1358, D19S433 and amelogenin loci <150 bp in size remained essentially intact and could be readily re-amplified (Supplementary Figure S1). Eventually, the control DNA 9947A-derived allelic peaks were eliminated by thermally and temporally extended autoclaving (128°C, 420 min; Table 1). Although control DNA 9947A-derived allelic peaks were not observed, non-allelic broad peaks were observed (Figure 1(a) and (b)). These peaks were also generated from the thermally and temporally extended autoclaved amplified STR products (negative control; Figure 1(d) and (e)). However, they were not observed for the thermally and temporally extended autoclaved allelic ladder (Figure 1(f) and (g)). Moreover, these artefacts were also observed in the no amplification controls (Supplementary Figure S2). To analyse the artefacts, autoclave experiments were also conducted using the AmpFℓSTR^® Yfiler^® PCR amplification kit. Similar non-allelic peaks were observed in the autoclaved samples, despite the use of a different kit (Supplementary Figure S3).

Table 1.

Effect of autoclaving for amplified STR product elimination.

Dried amplified STR product	Autoclaving
Dried amplified STR product	121°C			128°C
(µL)	20 min	300 min	420 min	20 min	300 min	420 min
10	+	+*	+*	+	+*	−*
1	+	+*	−*	+	+*	−*
0.1	+	+*	−*	+	−*	−*
0.01	+	+	−	+	−	−
0.001	+	−	−	+	−	−
0.0001	−	−	/	−	−	/
0.00001	−	/	/	−	/	/

*Non-allelic peaks were observed.

STR: short tandem repeat; +/−: remaining and eliminating allelic peaks; /: not examined.

Figure 1.

Electropherograms of short tandem repeat (STR) typing results. Amplified STR products were detected by capillary electrophoresis using a 3130xl Genetic Analyzer. The vertical axis indicates relative fluorescent units, and the horizontal axis identifies data-collection points. The template for each amplified STR product was: (a) 1 µL of the amplified STR product derived from 0.5 ng of control DNA 9947A (positive control); (b) the autoclaved (128°C, 420 min) amplified STR products (positive control); (c) the autoclaved and then subjected to UV irradiated (60 J/cm²) the amplified STR products (positive control); (d) 1 µL of the amplified STR products derived from ultrapure water (negative control); (e) the autoclaved amplified STR products (negative control); (f) 1 µL of the allelic ladder; and (g) the autoclaved allelic ladder. Non-allelic peaks were observed in (b) and (e). *Non-allelic peaks. Black vertical lines indicate the data collection points corresponding to 420 bp-sized DNA amplicons.

UV irradiation

To remove PCR-amplifiable DNA, we also tested the effectiveness of UV irradiation in a range from 10 to 60 J/cm². PCR-amplifiable DNA was not removed by UV irradiation (10–60 J/cm²) from the dried amplified STR products (0–10²-fold dilution). A UV dose-dependent effect was not observed. However, PCR-amplifiable DNA diluted 10³-fold, which was otherwise not removed by autoclaving under standard sterilisation conditions, was eliminated by 10 J/cm² of UV irradiation (Table 2).

Table 2.

Effect of UV irradiation for elimination of amplified STR products.

Dried amplified STR product	UV irradiation (J/cm²)
(µL)	10	20	30	40	50	60
10	+	+	+	+	+	+
1	+	+	+	+	+	+
0.1	+	+	+	+	+	+
0.01	−	−	−	−	−	−

UV: ultraviolet.

Combination of autoclaving and UV irradiation

To remove the non-allelic peaks derived from thermally and temporally extended autoclaving, the dried amplified STR products were autoclaved at 128°C for 420 min and then subjected to UV irradiation over the range 10–60 J/cm². After sequential autoclaving and UV irradiation, the allelic peaks were eliminated and the non-allelic peaks were absent from the mock contaminating amplified STR products (Figure 1(c)). UV irradiation reduced the non-allelic peaks in a dose-dependent manner (Supplementary Figure S4).

Comparison of DNA decontamination techniques for amplified STR product elimination

We verified the applicability of the autoclaving and UV irradiation combination approach by contaminating PCR tubes, latex gloves and cotton swabs with the amplified STR products. To compare the effectiveness with existing approaches, these mock contaminants were treated by not only the combination method but also bleach, ethanol and DNAZap™ solutions. Our results revealed that only the combination method was able to remove the amplified STR products contamination from all mock samples (Table 3).

Table 3.

Comparison of DNA decontamination techniques for amplified STR product elimination.

	DNA decontamination techniques
	AC & UV	Bleach	EtOH	DNAZap™
PCR tubes	−	+	+	+
Latex gloves	−	+	+	+
Cotton swabs	−	−	+	+

EtOH: ethanol; PCR: polymerase chain reaction.

Discussion

Effect of autoclaving

In this study, we evaluated the effectiveness of autoclaving and UV irradiation as DNA decontamination procedures for amplified STR products. Autoclaving under standard sterilisation conditions was insufficient to eliminate PCR-amplifiable DNA from the dried amplified STR products. The smaller amplicons of STR loci were detected after standard autoclaving because small amplicons may be resistant to degradation, a result consistent with previous studies.^4,13 Therefore, we tested a range of thermal and temporal parameters of autoclaving which could enhance DNA decontamination.^4,13 Gefrides et al.⁴ reported that autoclave decontamination (121°C, 120 min) could eliminate contaminating DNA from 10 µL of dried saliva. The amplified STR products were more difficult to remove than the contaminating cells; PCR-amplifiable DNA still remained after their temporally extended autoclaving (Table 1). In our study, the control DNA 9947A-derived peaks were eliminated by thermally and temporally extended autoclaving (128°C, 420 min). However, such a strong treatment generated artefacts to prevent proper STR analysis. The artificial peaks were observed in autoclaved amplified STR products derived from control DNA 9947A and ultrapure water (Figure 1(b) and (e)), but not in the autoclaved allelic ladder (Figure 1(g)). Thus, we consider that the artificial peaks were not dependent on the remaining template DNA because they were also detected in the no amplification controls with similar peak heights and data points. The artificial peaks had the following features: they were broad, similar to ‘dye blobs’^14,15 and observed at almost coincident data points. Moreover, similar artefacts were also observed in autoclaved samples derived from control DNA 007 and ultrapure water using the AmpFℓSTR^® Yfiler^® PCR amplification kit. The common point between the two commercial kits is using the same dye set (6-FAM™, VIC^®, NED™ and PET^®).^16,17 These results suggested that the artificial non-allelic peaks were derived from free dyes from dye-conjugated primer sets of the amplification kit. Therefore, the effectiveness of autoclaving alone for elimination of the amplified STR products is limited and imperfect.

Effect of UV irradiation

The effect of UV irradiation in reducing PCR-amplifiable DNA was insufficient to eliminate the contaminating amplified STR products (at 10²-fold dilution). Moreover, the parameter change of UV irradiation (10–60 J/cm²) had no clear effect on DNA degradation. UV irradiation acts on DNA through thymidine dimer and nick formation on DNA strands.³ Because UV affects exposed DNA on surfaces of materials, the degradation effects may be decreased in the presence of protective barriers such as cell and nuclear membranes, numerous proteins and/or DNA fragments. Therefore, we consider that UV irradiation is easy to use and effective for removal of contaminating superficial DNA in a short time frame, whereas it is not expected to increase the decontamination effect on DNA contaminants by extension of UV dose.

Combined effects of autoclaving and UV irradiation

Although PCR-amplifiable DNA of the amplified STR products was removed by the extended autoclaving, the powerful process resulted in artefactual peaks. If these peaks are non-allelic and derived from liberated fluorescent dye-conjugates, we considered that they may be removed using UV irradiation quenching. Our results (Figure 1(a)–(c)) demonstrated that the combination of autoclaving and UV irradiation was effective for the removal of the allelic and non-allelic peaks. Therefore, we examined forensic applications of the combination method using laboratory consumables and compared its effectiveness to other existing measures. As shown in Table 3, the only way to remove the amplified STR products, regardless of the carrier to which DNA was attached, was the combination approach. Our results suggest that this method may also be applied to other autoclave-possible instruments such as aluminium tube racks and stainless-steel tweezers used for pre-PCR operation. However, this combination procedure requires a lengthy processing time and has some possibility of providing incomplete effects, depending on the amounts of the contaminating amplified STR products.

Limitations of autoclaving and UV irradiation

The aim of this study was to establish a DNA decontamination procedure focusing on laboratory-related contamination. Causes of contamination can be variously categorised: case-related and/or -unrelated, interaction-related, production-related and investigation-related. Although such contamination may occur incidentally, investigation-related contamination can be controlled and should be prevented by effective countermeasures. Among DNA decontamination techniques in forensic science,^4,5,10–12 autoclaving and UV irradiation are prevalent and easy to use in laboratory settings. Our results indicate that the combination of autoclaving and UV irradiation is a more effective optional countermeasure for prevention of amplified STR product contamination in routine cleaning. However, some laboratory instruments and consumables cannot be subjected to autoclaving. Furthermore, because UV irradiation requires no ‘shadowing’ between the contaminating DNA and the irradiation source, it cannot guarantee the desired effect, depending on the quantities of contaminants or complicated instrument architecture. Therefore, forensic laboratories should implement comprehensive approaches, including utilisation of DNA-free consumables, physical separation of working areas between DNA amplification and other processes, individual processing of each evidentiary sample to minimise carry-over or cross-contamination and utilisation of elimination databases.¹⁸ Furthermore, establishing systematic mechanisms for identifying intra-laboratory cross- and/or carry-over contamination may be effective to ensure authentic evidentiary value of forensic DNA results.

Footnotes

Acknowledgements

The authors wish to thank Nanaka Noda and Masako Ohmura for their assistance in reviewing the manuscript.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors received no financial support for the research, authorship and/or publication of this article.

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