DNA extraction from human saliva deposited on skin and its use in forensic identification procedures

Saliva is usually deposited in bite marks found in many homicides, assault and other criminal cases. In the present study, saliva obtained from volunteers was deposited on skin and recovered for DNA extraction and typing in order to evaluate its usefulness for practical case investigation and discuss the contribution of forensic dentistry to saliva DNA typing. Twenty saliva samples were collected from different donors and used as suspects' samples. Five of these samples were randomly selected and deposited (250 microl) on arm skin. Saliva was collected from skin using the double swab technique. DNA from saliva and skin-deposited saliva samples was extracted by the phenol-chloroform method. DNA samples were amplified by PCR for DNA typing using a set of 15 STRs. The recovery of DNA from saliva deposited in the skin was 14 to 10 times lower than DNA quantity from saliva samples. DNA typing was demonstrated in 4 of 5 deposited saliva samples, the likelihood ratios estimated for these samples based on data of the Brazilian population were 1:11, 1:500, 1:159.140 and 1:153.700.123. Our results indicate that standardized procedures used for DNA collection and extraction from skin-deposited saliva can be used as a method to recover salivary DNA in criminal cases. However, it is important to observe that DNA recovery in forensic samples can be difficult. This study suggests that the analysis of saliva deposited on skin be incorporated into a criminal investigation since it may have great discriminatory power.


INTRODUCTION
The dental forensic field includes the identification of unknown human remains as part of a crime or disaster investigation.It also includes the analysis of stains and organic liquids from the buc-cal cavity or its contents, bite mark comparison, investigation of trauma and oral injuries such as personal injury cases, and dental malpractice 5 .In criminal investigations, one of the fundamental requirements is that the victim and aggressor be positively identified.
Forensic dentistry contributes to the forensic process by either a direct comparison of the deceased's dentition with that of known dental records or by enabling a profile of the individual regarding age at death, sex and possible racial ancestry in order to narrow down the search for a possible victim 5,8 .
Saliva may be found on victims of several violent crimes 29 .It has been shown that saliva can potentially be recovered and typed from bite marks [3][4][5]25 , cigarette butts, postage stamps, envelopes and other objects 9,22,24 . Stins of dried saliva are invisible, making its recognition and collection difficult 24 .However, the presence of saliva can be confirmed by an amylase assay 9 .
In a bite mark, tooth in combination with other mouthparts cause a mark on victims' skin or some object, which can be compared with the unique characteristics of a suspected biter's dentition by several methodologies 3,5,29 .There is no agreement among forensic dentists about the individuality (uniqueness) of the dentition or the behavior of human skin during biting.Nevertheless, distortions may modify, complicate or render impossible the interpretation of a bite mark.Different distortions can occur at different stages in the action of biting, such as dynamic and tissue distortions, and during examination and evidence recording 25 .
Besides the physical evidence present in a bite mark, there is biological evidence that can assist the investigation.During the biting process, saliva is deposited on the skin or object surface in enough amount to allow typing of the deoxyribonucleic acid (DNA) 23,25 .For this purpose, the bite mark area is swabbed using the standard bite mark operating procedures, and DNA can be extracted and analyzed 22,25 .
Polymerase chain reaction (PCR) allows replication of thousands of copies of a specific DNA sequence in vitro, enabling the study of small amounts of DNA 20 .The polymorphic repair of short tandem repeats (STR) mainly in small fragments also makes it possible to evaluate DNA from samples with a significant grade of degradation.A very important step from this procedure is to obtain DNA from this small amount 9,14,22,26 .
In the present study, saliva obtained from volunteers was deposited in the skin and recovered for DNA extraction and typing in order to evaluate its usefulness for practical case investigation and discuss the contribution of forensic dentistry to saliva DNA typing.

Samples
Saliva samples were collected from 20 nonrelated volunteers (11 male and 9 female, 24 to 47 years old) by asking them to spit into sterile plastic test containers (STLU Ind. e Com. de Artefatos Plásticos Ltda.-Me, São Paulo, SP, Brazil).Then, 1.5 ml of saliva sample from each donor were transferred to two 1.5 ml cryotubes (Axygen Scientific, Union City, CA, USA) and frozen at -20°C.The samples were collected at least 30 minutes after eating, drinking, smoking or kissing to minimize DNA contamination or PCR inhibitors 21 .
Five samples from the 20 donors were randomly chosen to deposit part of saliva on skin.For this purpose, saliva samples were deposited individually on one of the researchers' arm.Saliva was collected from this researcher to establish his DNA typing to be compared with the saliva donors' profiles.
The samples were obtained after informed consent from Brazilian donors, after the study protocol was approved by the Research Ethics Committee of the School of Dentistry, University of São Paulo (process number 41/01).

Experimental saliva deposition on the skin
Two hundred and fifty microliters from each saliva sample were placed on five delimited areas of the researcher's arm and skin areas were delimited with a marker pen.Harvey 8 (1976) estimated that 0.3 ml of saliva is deposited when making a bite mark; in this study, we used 0.25 ml to simulate the minor recuperation of saliva stain.After 10 minutes, the dried saliva was colleted from the skin by the double swab technique using a wet cotton swab (Vacutest Kima s.r.l., Arzergrande, PD, Italy) followed by a dry cotton swab 23 , and DNA was immediately extracted.
The allelic frequency of the Brazilian population 2,7,17 was used as reference and the match among samples was calculated on the basis of Likelihood Ratio (LR) 6 .

RESULTS
The DNA quantification of 20 saliva samples resulted in an average of 58.9 ± 43.2 µg.The amount of DNA of the 5 samples, named "A", "B", "C", "D" and "E", deposited on skin (250 µl) resulted in an average of 4.2 ± 0.9 µg.DNA recovery from skindeposited saliva was 14 to 10 times lower than DNA from saliva samples (p < 0.05).The DNA extracted from saliva samples collected from skin presented a A 260 /A 280 ratio of A = 1.4476;B = 1.8466;C = 2.0751; D = 2.2118 and E = 1.5737.The DNA typing of all saliva and control samples and skin swabs is shown in Table 1.STRs were amplified in 4 of 5 DNA samples extracted from saliva deposited on skin.
Despite the small number of amplified loci in the saliva samples collected from skin, it was possible to match the alleles of these loci in the 20 saliva samples, A with 5, B with 6, C with 7, E with 3 or 13 or 20.The likelihood ratios based on data from the Brazilian population were A 1:500; B 1:153.700.123;C 1:159.140; and E 1:11.

DISCUSSION
All the DNA profiles were obtained from the 20 samples, which could be compared with the other five samples deposited on skin.STRs were amplified in 4 out of the 5 DNA samples extracted from saliva deposited on skin, and that is probably related to the lower recovery of DNA from skindeposited saliva.
The single loci amplification was used in this research; however, the multiple loci amplification could also have been used.The Spanish and Portuguese Working Group of the International Society for Forensic Genetics (SPWG-ISFG), who develop guidelines for and discuss quality assurance and control program in forensic genetics, accepts manual or semiautomatic STR typing and single or multiple loci amplification 1 .In a multiplex system, it is likely that more loci would be amplified 22 , but this was not tested.
To control contamination between DNA samples, we prepared them in separate rooms, with UV germicidal lamps that damage any DNA left on exposed surfaces.The solutions were autoclaved, reagents were divided into aliquots and mixed prior to being divided into aliquots; DNA was added in another room and positive and negative controls were used.This procedure was very important to avoid false positives with PCR 11 .
Another relevant aspect occurs when the amount of evidence available is minimal 22 , because the DNA sample may not be enough to accomplish the DNA typing.However, in the present study, the DNA quantity from saliva deposited samples was sufficient for PCR amplification and typing.Nevertheless, in sample D no alleles were amplified.The A 260 /A 280 ratio of DNA extracted from saliva samples colleted from skin cannot justify sample D negative loci amplification.Perhaps, unknown PCR enzyme inhibitors could be present in saliva samples 26 , or salivary DNA was not extracted in enough amount to allow amplification.We have to consider that saliva may have microorganisms 4 that can be source of DNA that can be extracted and quantified.It was demonstrated that DNA recovery from the saliva deposited on skin was not proportionally equivalent to the DNA quantity found in the 20 saliva samples.Saliva deposition, salivary cell collection and DNA extraction procedures probably reduce the DNA quantity.
Despite the small number of amplified loci in the saliva samples collected from the skin, it was possible to match the alleles of these loci in the 20 saliva samples (A with 5, B with 6, C with 7, E with 3 or 13 or 20).The likelihood ratios based on data from the Brazilian population that varied from 1:11 to 1:159.140suggest that STR analysis in DNA samples from skin-deposited saliva can be used as strong evidence to assist the criminal process 22,26 by exclusion or inclusion of suspects.However, the solution of a forensic case requires other important studies such as the physical bite mark and other evidences found in the crime scene that should be well addressed by the investigators.
In this experimental study, it is important to consider that there is no contact between lips and teeth and skin, as happens in a real case, where buccal cells are impregnated on skin, which can increase cell quantity.In a real case, there is tissue invasion, with the possibility of mixing the victim's and the aggressor's DNA 27 .When there are mixtures, the contamination can be recognized during case investigation, comparing the victim's DNA and the DNA of the suspect.However, in this research, the results showed that DNA samples from skin-deposited saliva were not mixed with control DNA, corroborating the results from another similar previous study 26 .

CONCLUSION
Our results indicate that standardized procedures for DNA collection and extraction from skin-deposited saliva can be very useful to help solving criminal cases, since important data may be obtained using salivary DNA analysis.Therefore, the analysis of saliva deposited on skin can be incorporated into a criminal investigation and have great discriminatory power.