Technical noteComparison of Quantifiler® Trio and InnoQuant™ human DNA quantification kits for detection of DNA degradation in developed and aged fingerprints
Introduction
The modest objective of the work reported here was to compare the efficacy of the recently developed Quantifiler® Trio (Thermo Fisher Scientific, Oyster Point, CA, USA) and InnoQuant™ (InnoGenomics, New Orleans, LA, USA) kits for measuring the extent of degradation of DNA in forensic samples. Samples chosen for the comparative study included latent fingerprints that were first developed for enhancement of visualization and then aged in ambient conditions. As reference, DNA was also quantified in latent fingerprints that were not developed but still aged.
Both fingerprint examination and DNA profiling are widely used methods of identification. Application of two different identification techniques on one evidence sample can strengthen the validity of evidence [1]. Latent fingerprints are developed and compared with fingerprint databases as well as against fingerprints harvested from suspects and other individuals at crime scenes. Genomic DNA from fingerprints has been shown to yield successful short tandem repeat (STR) profiles, and mitochondrial DNA sequencing has been performed from palm prints on paper [2], [3]. Thus, analysis of DNA from fingerprints is valuable in resolving criminal cases.
A significant difficulty in processing these samples for both enhanced visualization and DNA analysis is that the fingerprint often contains low amounts of DNA. In outdoor crime scenes, exposure to excessive heat or cold as well as to humidity or aridity can preferentially degrade higher-molecular-weight DNA [2]. In addition, peculiar DNA damages are caused by UV radiation [4]. Environmental insults may result in allelic drop-outs, yielding partial DNA profiles, commonly observed when attempting amplification of low-template or degraded DNA with STR kits [5], [6], [7].
In forensic laboratories, samples containing small amounts of DNA may be stored for long periods of time. Assessment of degradation in DNA-containing samples is becoming increasingly commonplace [8]. Hence, it is now known that while environmental factors such as high/low temperature and humidity/aridity play major roles in degrading DNA, aging at ambient conditions can also incur severe degradation [4].
Traditional fingerprint-development techniques to enhance visualization for forensic examiners may be detrimental to the preservation of DNA within the fingerprint residue. For example, black powder may degrade DNA [2], [3], Crowles double stain and Hungarian red may reduce DNA amplification efficiency [9], and cyanoacrylate may reduce specific products of the polymerase chain reaction (PCR) [10]. Furthermore, airflow and exposure to UV radiation can degrade biological initiators of cyanoacrylate polymerization [11]. Thus, many traditional fingerprint-development techniques have the potential to inhibit downstream DNA analysis [2], [9], [11], [12]. In contrast, the columnar-thin-film (CTF) technique borrowed from nanotechnology involves the deposition of a 50–1000-nm-thick CTF conformally on a latent fingerprint [13], [14]. The CTF entombs the fingerprint residue and may thereby preserve DNA in the residue. As CTF deposition occurs in a low-pressure chamber the biological material may be altered or depleted [15]. However, recent study indicates that biological material such as blood and saliva are not degraded by the CTF deposition [16].
In order to overcome difficulties in typing degraded, inhibited, and low-template DNA, often referred as low-copy-number (LCN) DNA, amplification kits such as the AmpFlSTR® MiniFiler™ PCR Amplification kit have been developed [7]. With these kits which generate amplicons of less than 270 base pairs (bp), it is possible to amplify extracts that may contain degraded DNA as well as inhibitors of the polymerase chain reaction (PCR). The results obtained from partial DNA profiles can be combined with those obtained with miniSTRs for better discrimination. However, when samples contain very low amounts of DNA, amplification of extracts in duplicates can consume the samples, leaving no possibilities for opposing counsel to repeat the tests. Also, in forensic laboratories every amplification kit used on casework samples must be validated, which leads to budgetary constraints and loss of valuable time. Recent research has employed the use of a standardized degraded DNA sample to help in determining critical parameters for comparing different kits [17].
As mentioned at the beginning of this section, the main objective of this research was to compare the efficacy of the two recently developed DNA quantification systems: Quantifiler® Trio and InnoQuant™ kits. Both of these kits, which use a quantitative PCR (qPCR) assay, can assess the amount of human DNA and can also determine the level of DNA degradation, thus providing guidelines for more adequate downstream STR analysis [18], [19]. Most of the samples chosen for the comparative study were latent fingerprints that were either (i) developed using one of three different techniques and then aged in ambient conditions or (ii) undeveloped but still aged. Furthermore, in order to properly evaluate the differences in their performance, manually degraded samples were produced by controlled exposure of DNA standards to UV radiation.
The extent of degradation is determined by comparing the amount of a short strand of amplified DNA with the amount of a long strand of amplified DNA to obtain a DNA degradation ratio. Some researchers have shown that the use of one short DNA target and one long DNA target demonstrates a positive relationship between an increasing DNA degradation ratio and a loss in longer STR alleles [20], [21], [22], [23]. Others have implemented this technique with the use of an internal positive control or Y chromosome target [20], [21]. Different quantification kits available in the forensic community utilize different targets for the same assessment [18], [19], [24]. The use of different DNA targets in different qPCR systems may lead to different results for the sensitivity of DNA quantitation, the extent of DNA degradation, and the level of inhibitors present. The differences in the origin of these targets in the human genome may lead to slight differences in the values obtained by the quantification procedure. Since these quantification kits are used for comparison of the amounts of the short amplicon with that of the long amplicon, differences in the length of the long amplicon may lead to different degradation ratios.
Section snippets
DNA standards
Standard samples used for the manual-degradation study included 9947a, control DNA in the amplification kits from Life Technologies (Oyster Point, CA, USA), concentration 0.10 ng/μL and 2800 M (Promega Corporation, Madison, WI, USA), concentration 10 ng/μL. These two DNA standards with different concentrations, one low and other high, were chosen in order to examine the degradation patterns when DNA is present either as low-template DNA (such as in fingerprints) or in larger quantities (such as in
Samples degraded manually by UV exposure
Two DNA standards were subjected to manual degradation by defined exposure times to UV radiation. Fig. 1 presents the degradation ratio of DNA standard 9947a (concentration 0.10 ng/μL) plotted as a function of UV-exposure time (0–60 min), as measured by the Quantifiler® Trio and InnoQuant™ kits. Analogous data for the DNA standard 2800 M (concentration 10 ng/μL) are presented in Fig. 2. Even though the two kits used in this research share similarities in their methods for quantification of DNA,
Concluding remarks
This research explored the variation in DNA degradation as a result of different methods of fingerprint development and of aging, as determined by degradation ratios obtained from two recently available real-time PCR quantification kits. As fingerprints contain low amounts of DNA, the obtained results may also be applicable to other low-template DNA samples.
Reliability appeared to depend on the quantity of DNA and the degree of degradation in the sample. Low-template DNA was responsible for the
Funding
This study was supported by the Forensic Science Program of the Eberly College of Science as well as by the Charles Godfrey Binder Endowment, both at the Pennsylvania State University.
Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All samples were collected following the guidelines provided by the Institutional Review Board (IRB) and Institutional Biosafety Committee (IBC) of The Pennsylvania State University, University Park, Pennsylvania.
Conflict of interest
None of the authors have any financial conflict or disclosure to make regarding this study. The authors were not compensated financially for doing this research.
Acknowledgments
The authors are grateful for helpful suggestions from Steven B. Lee (San Jose State University), Robert C. Shaler (The Pennsylvania State University), Dawn Waltman and Jamie Brachold (Thermo Fisher Scientific, Inc.), Teresa Snyder-Leiby (SoftGenetics LLC), Gina Pineda (InnoGenomics), and Jaiprakash Shewale (Rowpar Pharmaceuticals, Inc.).
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