The Quality of mt-loop DNA Segment of Smokers and Nonsmokers in Lagos, Nigeria for Possible Use in Forensic Biology

Mitochondrial DNA (mtDNA) is a small circular DNA responsible for transmission of traits. In forensic biology, the high sensitivity of mtDNA analysis allows forensic scientists to obtain information from evidence associated with crime scene. This study was carried out to investigate the mtDNA segment of smokers and nonsmokers and to determine to what extent smoking affects the quality of the mtDNA in the sample population. Twenty five cigarette butts were obtained from a bar and twenty five samples were also obtained from saliva of non-smokers using swab stick. Mitochondrial DNA (mtDNA) was extracted from individual samples using zymo kit, spectrophotometer was used to check for the concentration and purity of the extracted mtDNA. Polymerase chain reaction (PCR) was carried out in a gradient thermocycler to ascertain the hypervariable region of the mtDNA using the following primer sequence. Agarose gel electrophoresis was carried out to know the amplicon size using 100 base pair of ladder. The DNA purity on saliva extract for non-smokers was found to be higher (A260/280 2.06 1.82) than the purity of saliva from smokers (A260/280 1.82-1.0). The concentration of DNA found on the saliva traces from non-smokers was higher (26.2 3.0 ng/μl) than those extracts from smokers (26.2 2.23ng/μl). DNA bands obtained from agarose gel electrophoresis showed amplification of the hypervariable region of mtDNA size ranges from 295-300 base pair (bp). This study showed that the hypervariable region of the mtDNA of both smokers and non-smokers have the same range of nucleotide base pair.


Introduction
Forensic identification is an effort to help law enforcement in determining a person's identity. Personal identity is often a problem in criminal cases, civil cases, death without identity, and mass disasters 1 . Personal identification is defined as the act of establishing the identity of an individual. It arises in natural mass disasters like earth quakes, tsunamis, landslides, floods etc., and in manmade disasters such as terrorist attacks, bomb blasts, mass murders, and in cases when the body is highly decomposed or dismembered to deliberately conceal the identity of the individual 2 .
There are several types of personal identification in forensic biology such as DNA analysis, Friction Ridge Analysis, Forensic Odontology, Biometric detection 3,4 . Deoxyribonucleic acid(DNA) analysis of evidential material is a powerful tool for linking an individual to a victim or a crime scene, DNA is a molecule that carries genetic material 5 which has been successfully extracted from many biological sources such as blood, semen, saliva, skin cells and used for different scientific analysis 6 .
Human DNA is found in every cell except erythrocytes 7,8 . DNA is unique to every individual and DNA typing methodologies are continuously subjected to scientific analysis. It is important because it contains genetic information which can be used in disease detection, profiling the paternity of a child, examining the biological mother of a child 9 and to identify individuals by the characteristics of their DNA for resolution of criminal cases.
The human genome contains about 3 billion base pair which is important in carrying genetic information that helps to identify an individual 10 . In human cells, most DNA is found in a compartment within the cell called a nucleus and it is known as nuclear DNA 11 . In addition to nuclear DNA, a small amount of DNA in humans and other complex organisms can also be found in Hypervariable region 1 (HVR-I), Hypervariable Region 2 (HVR-II) and Hypervariable Region 3 (HVR-III). HVR1 is considered a low resolution region and HVR2 is considered a high resolution Changes or repeats in the hypervariable region are highly polymorphic and these regions are useful in human mitochondrial genealogical DNA testing and widely used as a tool in many fields including evolutionary anthropology and population history, medical genetics, genetic genealogy, and forensic science.
Maternal inheritance of mtDNA allow scientists to compare the mtDNA profile of a set of remains to that of reference samples from individuals such as the mother, brother(s), sister(s) or any other maternal related individuals of a missing person. These samples should have the same mtDNA profiles because all maternal relatives inherit the same mtDNA. Since mtDNA is maternally inherited and multiple individuals can have the same mtDNA type, unique identifications are not possible using mtDNA analyses. However, mtDNA is an excellent technique to use for obtaining information in cases where nuclear DNA analysis is not feasible.
It is generally accepted that about 10% of genome is genetically relevant and the other non-coding region, which represents about 90% of the human genome, has part of it as repetitive sequence 13 . The repetitive sequence forms number of blocks that varies considerably among unrelated individual.
These repetitive sequences are majorly categorized into mini and micro satellites 14,15 . Satellite repeats possess extreme diversity in their monomer size, nucleotide sequence, complexity, genomic distribution, and abundance even in closely related species 17,18 . Microsatellites are widely used for DNA profiling (genetic fingerprinting) in forensics biology such as in crime scene analysis and also in tissues transplant in patients. They are also widely used in kinship analysis (most commonly in paternity testing). 18  DNA has been used as a unique investigation material for forensic purposes 8 . DNA amplification methods have been developed to quantify DNA since mid-1980s 20 . This method is referred to polymerase chain reaction (PCR).
The methodology of DNA typing is majorly coupled with the sensitive polymerase chain reaction, and it has ever since been used for research based analysis and legal scrutiny individualization.
Every individual has a distinct DNA and methodologies of DNA typing are continually exposed to scientific and legal scrutiny 21 . DNA traces can be found in body fluid; like blood, saliva, teeth, semen, vaginal secretion, bones, hair and perspiration 22 . DNA Forensic samples can be composed of DNA from more than one individual, making the typing results more complex 23 . Forensic materials that contain degraded DNA or minute amount of DNA may not yield a STR result 24 .
The degradation could be as a result of environmental conditions, hence the need to investigate the quality and purity of DNA; cigarette butt remains a very quality source where we can get DNA because of the dried saliva of the smoker on it. DNA samples recovered from dried saliva in a cigarette butt left at crime scene are frequently exposed to detrimental environmental factors (such as light, heat, decomposition of bacteria among others) before they are collected, which will affect the results of the analysis that will be carried out on them 25 .
The possibility of obtaining a reliable sample that could be analysed as evidence becomes a huge challenge to the forensic odonatologists 26 . Traces of saliva have been known to be mainly found at cigarette butt which takes 18 months to 10 years to decompose, depending on condition. These traces of saliva from the cigarettes butts are useful in forensic practice 25 .
Currently, about 1.2 billion people globally smoke tobacco whenever they are distressed and some other times get addicted. The number of smokers worldwide has increased to 1.1 billion in 2019, with tobacco smoking causing 7.7 million deaths including 1 in 5 deaths in males worldwide 27 .
In Nigeria, about 16100 tobacco-related deaths occur annually 28 . It is likely that these numbers may be grossly underestimated because of weak surveillance systems. In addition, 5.6% (4.7 million) of Nigerian adults currently use a tobacco product and 3.9% (3.1 million) adults are current tobacco smokers 29 . Of greater concern is tobacco smoking by children and adolescents where 25000 Nigerian children (aged 10-14 years) smoke cigarettes each day.
Cigarettes are affordable for young people in Nigeria because they are still being J, Minari sold in single sticks, despite the provision of Article 16 of the Framework Convention on Tobacco Control to which Nigeria is a signatory 30,31 .
In forensic practice, the most common carriers of saliva traces are cigarette butts, chewing gums, postage stamps and envelopes. Saliva is a complex biological fluid secreted by acinar cells of the major and minor salivary glands. It is an indicator of various plasma constituents. In recent years, its role as a diagnostic and forensic tool is being increasingly researched upon and evaluated. Besides maintaining the homeostasis of oral structures such as tooth integrity. It also plays a critical role in genomics, proteomics, metabolomics, and bioinformatics. It is an important discriminating element in forensic biology, acting as an indicator of salivary gland conditions and toxicological and drug monitoring 32,33,34 .
In a crime scene, smokers leave their cigarette butts unknowingly, sometimes could help forensic scientists to clarify identities of individuals involved in the crime by extraction of the DNA in the leftover saliva. In other words, these could lead to misinterpretation of the events of the crime scene and compromise the results from the event. Also there are some methods of DNA extraction for trace DNA such as from butts but their DNA yield and quality were not as good to get a good genotyping results. Examination of saliva traces left on cigarette butts as evidences are complicated due to the availability of the biological material in trace amounts for analysis and its rapid degradation due to extreme effects of environmental factors. There is a growing need to perform trace analyses such as DNA salivary analyses on cigarette butts found at a crime scene for identification purpose. Hence, this study will compare the DNA quality and quantity in cigarette butts recovered from smokers and analyzed with specific primers to determine its yield and quality. The positivity of the result will determine its use in forensic studies in Nigeria.
The aim of this study is to assess the quality of MT-loop DNA segment of smokers and nonsmokers for possible use in forensic biology. The specific objectives of this study were to determine the quantity and quality of DNA extracted from cigarette butts and swab stick, determine the concentration and purity of DNA using Nano-drop Spectrophotometer and evaluate the amplification of mt-DNA of smokers and nonsmokers using PCR and primer.

Sample collection
Twenty-five samples of used cigarette butts were provided by smokers from a bar at University Road Lagos Mainland Akoka, Yaba, Lagos and twenty-five samples were collected from buccal cells of non-smokers (students) from faculty of Science, University of Lagos using swab stick. The samples were aseptically transferred into zipped poly and red cap bottles then stored at -20 o C till further experiments.

Ethics approval
Application for ethics approval for the research was obtained. The Ethical Approval Number is CMUL HREC, Registration Number: NHREC/19/08/2019B.

DNA extraction
Each of the twenty-five (25) butts was cut in Eppendorf tube containing 500ul of genomic lysis buffer. The samples were vortexed for 4-6 secs and centrifuged at 10, 000 rpm for one (1) minute to dislodge other materials. It was allowed to stand at room temperature for 5-10 minutes. The suspension was transferred to a Zymo-Spin TM column in a collection tube and centrifuged for a minute. The residue was discarded from the flow through and the supernatant in the Zymo-Spin TM column was transferred to a new collection tube. DNA prewash buffer of about 200ul was added to the spin column and then centrifuged for a minute.
Following this, 500ul of genomic DNA wash buffer was added to the spin column and centrifuged again for another one (1) minute. The suspension in the spin column was transferred to a clean micro centrifuge tube and 100ul DNA elution buffer was added to it. This mixture was incubated for 2-5 minutes at room temperature. It was then centrifuged at 12000rpm for 30 seconds to elute the DNA.

DNA quantitative analysis
The DNA quantity was measured using the Nano-drop spectrophotometric analyzer with a wavelength of 260 and 280. The DNA quantity of smokers' ranges from 3.0-56.7ng/l and purity level ranges from 1.0-1.82 while that of the non-smokers ranges from 2.23-52.6ng/l and the purity level ranges from 1.14-J, Minari 2.06 respectively. The absorbance quotient (OD 260 /OD 280 ) provides an estimate of DNA purity. An absorbance quotient ratio < 2.0 was considered to be purified DNA. A ratio of < 1.8 is indicative of protein contamination, whereas a ratio > 2.0 indicates DNA contamination.

Agarose gel electrophoresis
The integrity of genomic DNA was tested by resolving DNA extract on a 1% agarose gel by electrophoresis followed by visualization with 8ul ethidium bromide staining. Each DNA sample was graded according to the electrophoretic migration of sample DNA compared with a known molecular weight marker 35 . PCR amplification products (10 μl) were subjected to electrophoresis (Bio-Rad) on 1% agarose gel in 0.5× Tris-Borate-EDTA buffer at 70 V for 1hr and stained with ethidium bromide (Himedia), and images were obtained in gel documentation (G-Box; Syngene, Cambridge, UK) systems.

Polymerase chain reaction (PCR)
DNA extracts for the PCR-based assays was assessed by amplifying the mtDNA D-loop region, which was amplified by PCR using primers forHV1 and

SPSS statistical analysis
Statistical analysis was by one way analysis of variance (ANOVA) using SPSS 20.0 version for the analysis on DNA isolation, P value lesser than (<) 0.05 was considered significant.

Results
The results from this study showed that the purity and concentration of DNA extracted from saliva traces on smoked cigarette butt and swab stick of nonsmoked to have variation among various sample as shown in Table 1 and 2 below. Table 1 Figure 1 shows the migration of genomic DNA band from negative anode to positive cathode in agarose gel electrophoresis. However, it was observed that some genomic DNA samples were stuck in the well.

Discussion
Saliva is a complex biological fluid secreted by the acinar cells of the major and minor salivary glands. It is an indicator of various plasma constituents. In recent years, its role as a diagnostic and forensic tool is being increasingly researched upon and evaluated 36  inheritance. mtDNA typing has become routine in forensic biology and it is used to analyze old bones, cheek cells, teeth, hair shafts and other biological samples where nuclear DNA content is low 38 .
In this study, the purity of DNA obtained from non-smoker group showed What was observed in the agarose gel electrophoresis for genomic DNA indicates that there was DNA in the extracted samples, and the bands showed that the DNA was good enough and of high quality. The research then proceeded to the next level of the analysis using the polymerase chain reaction (PCR). The results obtained showed the amplification of mtDNA. The band seen in the region of 295-300 base pair corresponding to molecular weight marker (l) of 100 base pair among non-smokers and smokers suggests that the mtDNA was amplified and it could be of relevance for forensic analysis. mtDNA has been shown to be more durable than nuclear DNA (nDNA) using electron microscopy. The results are attributed to its high number of copies per cell and the tolerance to extreme environmental circumstances, as well as other unique characteristics such as haploid maternal inheritance, high levels of variety, absence of recombination, lack of introns and histones and due to rapid evolution 43 . This study also found that both smokers and non-smokers have the same nucleotide base pair (bp) ranges in the amplified hypervariable region of their mtDNA.

Conclusions
This study was carried out to determine the reliability of mtDNA in forensic biology and the effect of smoking on this reliability. The results of the initial extraction showed that DNA purities of non-smokers were higher than those of smokers, suggesting overall negative effects of smoking on DNA purity.
However, this had no effect on the amplifications of the mtDNA during PCR analysis since the DNA of both smokers and non-smokers showed amplification at almost the same range of base pair (bp). The findings of the study showed clearly that smoking had no discernable effect on the stability of mtDNA amplification. This suggests that environmental influences have no effect on mtDNA function. This study revealed that the amplified region of mtDNA can be used for various forensic studies. However, more research at the genomic level is needed to understand the overall variation in the region by sequencing it, which will aid in a precise identification of an individual.