With the increasing rate of violence, crimes and terrorism with advancement of weapons used, proper human identification is required for legal and humanitarian reasons which is considered as a challenging field of study and research in forensic science. In this regard, many studies have used the dental pulp for sex determination (Veeraraghavan et al., 2010; Zapico & Ubelaker, 2013; Nogami et al., 2008; Nayar et al., 2014; Naik et al., 2012).
A single tooth may be the sole evidence presented in the scene and being the most durable tissue enclosing a rich vascular network; that is why the tooth pulp was chosen for sex determination in the present study.
Decontamination procedure which was applied in the present study, did not include any bleaching agents or detergents, in contrast to that were applied in the previous studies (Sweet & Hildebrand, 1998; Gaytmenn & Sweet, 2003b; Kemp & Smith, 2005; Marjanovic et al., 2007; Alakoc & Aka, 2009).
(Higgins et al., 2013), proved that bleaching agents decreased the amount of DNA yield in comparison to non- bleach treated teeth. Therefore, in the present work we did not use this method of decontamination to avoid destruction of DNA or dissolution of the pulp.
Actually there were no cases of external DNA contamination encountered in this study, as the androgen receptor gene was successfully amplified in all the samples. This denotes the effectiveness of the used decontamination method.
To access the dental DNA, many authors crushed the whole tooth (Sweet & Hildebrand, 1998; Sivagami et al., 2000; Meyer et al., 2000; Baker et al., 2001; Rubio et al., 2009), while others preferred sectioning the tooth (Gaytmenn & Sweet, 2003b; Murakami et al., 2000; Shiroma et al., 2004; PreseEki et al., 2000). The main drawback of the crushing method is that it completely destroys the teeth which is hindering further investigations. In sectioning the teeth, the access to the pulp chamber, by clear fracture, needs an experienced dentist. It is also difficult to perform teeth sectioning in case of pulp retraction (Tilotta et al., 2010).
In agreement with (Tilotta et al., 2010), the pulp was successfully extirpated in the current study by a standard endodontic access. The main advantage of this method is being conservative, so the tooth can be used for further investigation. It is also performed under air and water spray to prevent heating, whereas crushing or sectioning produces heat which may damage the genetic material (Tilotta et al., 2010). However, the endodontic access method utilized in this study required the need for a skilled dentist where the pulp was extracted with sufficient quality and quantity even from elder teeth.
The DNA extraction protocol used in the current study was based on solid phase extraction using silica- based column in contrary to other studies, where researchers used the classical organic extraction (phenol-chloroform method) (Sweet & Hildebrand, 1998; Murakami et al., 2000; Michaelis et al., 2008; Bulter, 2012).
The phenol chloroform method had many disadvantages including time consumption, difficulty and demanding of large quantity of samples which is not always available in real forensic investigations. Moreover, it requires the sample to be transferred between multiple tubes and this increases the risk of contamination. Also, handling of hazardous chemicals in this method poses a risk on the researchers (Michaelis et al., 2008; Bulter, 2012).
Utilizing the silica- based spin columns resulted in successful extraction of DNA from dental pulp samples with the ability to determine the desired elution volume that allowed maximum concentration of DNA, which was chosen to be 40 μl in the present study.
Sex determination was successfully achieved in all the studied samples with the use of SRY and DYS14 genes. Moreover, the age of participants had no impact on the potentials for sex determination.
Choosing SRY gene was based on its successful detection in different samples e.g.: blood, saliva, teeth and even from epithelial cells adhered to the bristles of tooth brushes (Vikram Simha Reddy et al., 2011).
Following several studies that showed deletions in the amelogenin gene on Y chromosome and subsequently the wrong identification of males as females, there was a definite need to use other reliable sex determining genetic markers (Kastelic et al., 2009; Kashyap et al., 2006; Chang & Burgoyne, 2003; Thangaraj et al., 2002; Drobnic, 2006; Laverde, 2013).
The multi-copy DYS14 gene was not conventionally used for sex determination in forensic cases, therefore, it was used in this study to verify its efficacy for sex determination from the tooth pulp. (Nakata et al., 2010) and (Blagodatskikh et al., 2010) previously reported its success in identifying sex from prenatal and blood samples. Additionally, (Gomaa & Sheta, 2013) have used DYS14 gene to identify male DNA in a mixture of male and female blood samples in varying proportions.
To compare between the sensitivity of DYS14 gene and SRY gene for sex determination using dental pulp samples, real time PCR would be adventitious in measuring the quantity of the amplified DNA bands. Thus, real time PCR is recommended for future studies to verify the contradictory findings reported by (Blagodatskikh et al., 2010) who documented that SRY gene showed ten times higher sensitivity than DYS14 gene. Whereas, other contradictory studies recommended DYS14 marker for the diagnosis of fetal gender from maternal plasma, and this recommendation was based on their findings which showed higher sensitivity of DYS14 over the SRY gene (Fernández-Martínez et al., 2012; Jacob et al., 2015; Khorshid et al., 2013). Hence, using conventional PCR in the present study provided 100% success in sex determination from all dental pulp samples with no false negative or positive results.