An introduction to the single cell gel electrophoresis assay: a technique resolving issues in forensic science

In death investigation, accurate time since death (TSD) estimation is a crucial task (Haglund 2002). Physical and biochemical changes which occur in the body after death can be helpful together to estimate the time of death (Donaldson et al. 2013). In the present scenario, various techniques have been used for estimating TSD. But precise estimation of TSD becomes difficult in the presence of numerous influencing factors (cadaveric or environmental origin) which can influence the ‘normal’ rate of postmortem changes, simultaneously. Generally, TSD can be estimated by two different methods (Pounder 1995). First one is Concurrence-based methodology. This method is based on the occurrence of a known event, which took place at a known time, with the occurrence of death, which took place at an unknown time. For example determining the age of manufacturing of clothing found on a body, tree ring development, dates on personal effects, etc. Concurrence based methods rely on both evidence associated with the body and anamnestic evidence such as the deceased’s normal pattern of movements (Pounder 1995).

The second technique is rate of change methodology. This method is to measure some aspect of evidence, directly associated with the body, which changes at a known or predictable rate and is started or stopped at the time of death. This method includes body temperature, tissue decomposition, insect succession and bone weathering (Pounder 1995).

By using the rate-of-change methodology, early TSD can be estimated on the basis of observational changes occur in human body after the death. The cooling of the body after death (algor mortis), the gradual stiffening of the body (rigor mortis) and the fixed pooling of the blood resulting in discoloration of the lower portions of the body (livor mortis) can be easily assessed with minimal instrumentation (Clark et al. 1997).

These Postmortem changes can roughly estimate the TSD but are unable to provide accurate estimation. Furthermore, these postmortem changes are very complex and influenced by the different internal and external factors (Swift 2006).

All of the above mentioned methods are effective but are insufficient to achieve the precise conclusion regarding to the TSD estimation. Instead of independently, these observational changes can collectively predict well, but all these changes are influenced by cadaveric or environmental origin, so it may vary from case to case and person to person. Therefore, accurate TSD estimation is not possible with these methods. Thus more accurate and reliable method for TSD estimation is urgently needed.

In spite of the tremendous advancement in forensics and life sciences over the past few decades, especially through DNA analysis, TSD estimation is still lacking behind. DNA is a genetic material and present inside a nucleus of the cell, therefore external and internal factors would not be able to affect them easily. Hence with the help of DNA degradation analysis we could reach to the more accurate estimation of the TSD.

Previous studies have shown that DNA degradation and repair is mutual interchangeable biological processes occurring in the cells of living human bodies (Reddy 2009; Clancy 2008). When any xenobiotic substance gets internalized into the cellular system, it may interact with genetic material such as DNA. When they interact with DNA, conformational changes, single or double strand breaks, formation of alkali labile sites may occur (Clancy 2008). When these changes are occurring in DNA, repair mechanism starts functioning immediately which is mainly regulating cell division process. These cell division processes stops in the cells, after death, then DNA repair pathway are impeded and DNA degradation process becomes un-repairable (Clancy 2008). In this way, DNA degradation increase with prolongation of time of death and thus, this could be a good molecular clock for TSD estimation. In this order, Xiong et al. (2010) have quantified the rate of DNA degradation in tissue cells with the prolongation of postmortem interval (PMI). This study revealed a positive linear correlation between the rate of DNA degradation and postmortem interval (Xiong et al. 2010). In addition, forensic researchers have used a variety of techniques in this concern with different tissues as sources of samples which demonstrated the positive correlation between DNA degradation and postmortem interval (Xiong et al. 2010; Lin et al. 2000; Chen et al. 2005; Luo et al. 2006; Zhen et al. 2006; Hao et al. 2007; Gomaa et al. 2013; El-Harouny et al. 2008). In this order, forensic scientists have made an attempt to use flow cytometry technique for estimating postmortem interval through the assessment of DNA degradation (Williams et al. 2015; Cordeiro et al. 2015). Additionally, they have found good correlations between TSD and DNA degradation measured using flow cytometry. But limitations to the procedure as it was originally proposed have proven difficult to overcome for forensic samples. Flow cytometry requires a suspension for staining and analysis. This could make scientists difficult to analyze solid tissue without extensive manipulation. Additionally, this assay measures total DNA but cannot be able to differentiate between intact human DNA and bacterial or fungal DNA. Several modifications to flow cytometry have been made due to which it become more specific and sensitive (Goddard et al. 2006), but the approaches have not extended the general acceptance. Therefore, this technique is not commonly used for forensic purposes. Another point is, this instrument is not easily available, due to high cost. For the forensic purposes we need a low cost, rapid, reliable, easily available and accessible technique that can be performed easily in the laboratory. At this instant, single cell gel electrophoresis (SCGE) or the Comet assay is the most appropriate method for TSD estimation via DNA damage assessment.

This technique was first introduced by Ostling and Johnson (1984), using a microgel electrophoresis technique to quantify DNA damage in cells. This technique is also known as “single-cell gel electrophoresis” which is famous by the name of “Comet assay” due to comet like appearance of degraded DNA in result (Fig. 1). They have used this technique in the neutral conditions that only allow the detection of double-stranded DNA breaks. Later on, NP Singh et al. (1998) have modified this technique as to assess the DNA damage in alkaline condition. The single cell gel electrophoresis (SCGE)/Comet assay combines the simplicity of biochemical techniques for detecting DNA single strand breaks (frank strand breaks and incomplete excision repair sites), alkali-labile sites and cross-linking with the single cell approach typical of cytogenetic assays. This is a simple, inexpensive, and sensitive technique to test for DNA damage.

Furthermore, Miteva et al. (2009) exhibited the forensic application of SCGE technique in sexual assault cases. This study revealed that SCGE help to measure the kinetics of sperm DNA degradation which help to estimate the time since deposition of sperm or time of ejaculation of sperm. With the help of this study, Miteva et al. (2009) elucidated that SCGE could be a valuable technique which may aid to the forensic investigation especially in the sexual assault cases. This study accurately measured the time since deposition/ejaculation of sperm qualitatively and quantitatively both.

Therefore, with the help of this paper an attempt was made to introduce SCGE assay, with its descriptive methodology and its role in the field of Forensic Science and also discuss about its future perspective.

Existing literatures have demonstrated that Single Cell Gel Electrophoresis assay play an important role in estimation of time of sperm ejaculation in rape and other sexual assaults cases by analyzing degradation of DNA content in sperm cells (Miteva et al. 2009). In this study, results revealed that DNA degradation rate was increased with time since deposition of stain. In addition, some studies have reported that SCGE is applicable to evaluate postmortem cell death processes, specifically nuclear DNA fragmentation (Xiong et al. 2010; Lin et al. 2000; Chen et al. 2005; Luo et al. 2006; Zhen et al. 2006; Hao et al. 2007; Gomaa et al. 2013; El-Harouny et al. 2008; Johnson and Ferris 2002). Among these studies, Johnson and Ferris (2002) shown that upon the death of an organism, internal nucleases contained within the cells caused degradation of chromosomal DNA into increasingly smaller fragments over time, and when these fragments were isolated and visualized, the fragmentation remarkably proved to be measurable and quantifiable of postmortem interval (Johnson and Ferris 2002). Moreover, some of these studies exhibited the strong statistical correlation between Comet assay parameters and postmortem interval/time since death estimation (Lin et al. 2000; Chen et al. 2005; Luo et al. 2006; Zhen et al. 2006; Hao et al. 2007; Gomaa et al. 2013; El-Harouny et al. 2008). Among these studies, Chen et al. (2005) reported that DNA degradation from heart, liver and kidney could be a hallmark for early time since death estimation. Rate of DNA degradation in first 6 h after death exhibited a linear correlation with postmortem interval. Similar findings also observed by Luo et al. (2006) and Zhen et al. (2006) who stated that tail length of DNA (indication of DNA degradation) increased with prolongation of PMI. In addition, Hao et al. (2007), El- Harouny et al. (2008) and Gomaa et al. (2013) have also demonstrated that the Comet assay is an appropriate method for early PMI estimation as evident by comet assay parameters (OTM, tail length). Apart from applying in PMI estimation and time since deposition estimation, this assay can also be used for environmental degradation assessment of biological sample & its repair (Lehmann 2003; Hall et al. 2004; Hall et al. 2014; Nelson et al. 2009). Some of these studies have successfully developed DNA repair systems (direct reversal by photolyase and single strand break/gap repair) which help to repair the DNA of degraded samples for certain extent (Lehmann 2003; Hall et al. 2004; Hall et al. 2014). In addition, instead of developing new DNA repair system, some of the studies have used commercially available repair enzyme (fpg, Endo III) to resolve the issue of degradation of biological sample (Nelson et al. 2009). In this case, advantage of Comet assay is that DNA repair enzyme can be incorporated in the procedure easily just before the electrophoresis step. In this way, Comet assay not only helps to measure DNA damage but DNA repair can also be measured.

Last two decade SCGE assay has been developed as an internationally recognized method to detect DNA damage or degradation in various animals and human cell types. Although it is believed that, the SCGE assay is still growing in use and has high potential for clinical, Toxicological and cancer research, yet in forensic science it is still new and latest.

The above mentioned studies (Miteva et al. 2009; Johnson and Ferris 2002) have successfully demonstrated that SCGE could play an important role in the field of Forensic Medicine but not frequently used. Apart from time since death estimation, SCGE can also be useful to repair slightly degraded biological samples, estimation of time since deposition of blood, semen, saliva, assessment of adverse effect of hazardous chemical on human (toxicovigilance) and biological samples.

In conclusion, from this article an attempt was made to open a new research avenue and awareness to estimate early post mortem interval using SCGE assay. Although SCGE assay has its some advantages and disadvantages, yet some modifications made this assay useful, reliable and consistent for variety of forensic applications.

In the forensic point of view author suggested that SCGE assay is a versatile technique which could be helpful to resolve issues like DNA degradation, time since death estimation, genotoxicity assessment of hazardous chemicals on biological sample. Introducing SCGE in forensic science will open a new avenue in the field of forensic research & innovation.