DNA quantification is a crucial process in molecular biology that involves determining the concentration of DNA in a given sample. Accurate quantification is essential for various applications including PCR, DNA sequencing, and forensic analysis. There are several methods employed for DNA quantification some of which include Spectrophotometry, Fluorometry, Real-Time Polymerase Chain Reaction (qPCR), Gel Electrophoresis, and Digital PCR (dPCR). These methods offer a range of options for DNA quantification, each with its own strengths and limitations. The choice of method depends on factors such as the type of sample, the required sensitivity, and the available equipment.

The buccal cavity stands as a readily accessible reservoir of biological material, indispensable in genetic inquiries. It presents an easily attainable avenue for biological sample acquisition, offering a non-invasive and cost-effective alternative to other sources like blood [2, 3]. Buccal swabs have garnered extensive utilization across a spectrum of research endeavors encompassing kinship analysis [4], forensic applications within law enforcement [4], antibody serostatus assays [5], rumen microbiota analysis [5], and HLA typing in HLA disease association studies [6], among a myriad of other applications. In stark contrast to venipuncture, which entails certain drawbacks including the prerequisite of a skilled phlebotomist for blood sample extraction and the inconvenience imposed on study participants by necessitated travel to designated collection sites, buccal epithelial cells prove adept at yielding human genomic DNA. Consequently, this approach engenders heightened participant engagement. Moreover, autonomous collection of buccal specimens has exhibited augmentation in recruitment outcomes, particularly in geographically remote or widely dispersed populations, establishing it as a superior strategy for procuring DNA samples from young children [7]. Notably, both passive drooling and swabbing for saliva samples, alongside the retrieval of buccal samples through swabs or brushes, stand out as preeminent means of securing oral samples [3].

While swabs have proven invaluable for sample acquisition, they have also been shown to yield suboptimal DNA quantities in downstream applications. The employment of wound fiber swabs as sample collection instruments emerges as a cost-effective, user-friendly, and easily transportable alternative. A prevalent swab design entails a wound configuration, with diminutive fibers (typically cotton) wound around a central shaft [33]. However, studies demonstrate a limitation associated with cotton swabs, manifesting in the tendency to introduce cotton fibers and other contaminants into the reaction mixture, potentially acting as inhibitors in subsequent applications such as Polymerase Chain Reaction (PCR) [8].

Human exposure to an array of genotoxic agents in our surroundings, food, and even endogenously produced electrophiles necessitates attention. These reactive chemicals have the potential to inflict damage upon our DNA, culminating in the formation of covalent modifications termed adducts [9]. Consequently, impurities within DNA samples wield considerable influence, imperiling the accuracy and reliability of experimental endeavors. These impurities harbor the capacity to compromise the integrity of DNA by diminishing its purity, in addition to inducing enzyme denaturation, thereby impeding further downstream analyses [10].

While swabs are meticulously designed to facilitate DNA sample collection from surfaces, an intriguing observation has surfaced. Subsequent DNA extraction from swabs often yields quantities significantly smaller than the theoretically maximum amount of DNA attainable (Bruijns et al., 2018). This phenomenon finds elucidation in the chemical interactions between DNA molecules and the functional groups on the swab surface. In specific scenarios, such as forensic touch samples, only minute quantities of DNA are present, resulting in inadequate DNA for other downstream molecular applications [8].

While buccal swabs provide a simple and noninvasive way to collect DNA, the quality and purity of extracted DNA often falls short of ideals for downstream testing. The A260/280 ratio is generally used to determine protein contamination of a nucleic acid sample. The aromatic proteins have a strong UV absorbance at 280 nm. For pure RNA and DNA, A260/280 ratios should be somewhere around 2.1 and 1.8, respectively. A lower ratio indicates the sample is protein contaminated. The presence of protein contamination may have an effect on downstream applications that use the nucleic acid samples. The A260/230 ratio indicates the presence of organic contaminants, such as (but not limited to): phenol, TRIzol, chaotropic salts and other aromatic compounds. Samples with 260/230 ratios below 1.8 are considered to have a significant amount of these contaminants that will interfere with downstream applications. This is especially true for reverse transcription. In a pure sample, the A260/230 should be close to 2.0. Previous studies using spectrophotometric analysis of DNA from buccal cells have reported A260/280 ratios as low as 1.3 and A260/230 ratios below 0.5, indicating significant protein and salt contamination [11]. Common issues include food residues, chemicals used during extraction, and mouthwash solutions retained during inefficient swishing protocols. These impurities can directly inhibit enzymes like polymerases or interfere with quantification to give false purity readings [12]. This necessitates optimization of pre-sampling preparation, collection materials, and extraction methods to isolate DNA of adequate purity from buccal sources. Achieving this optimization will facilitate more accurate DNA quantification and successful utilization in sensitive downstream genomics applications. Gender differences in DNA yields and purity from buccal swabs have been reported in some studies, but findings are inconsistent. For example, van Wieren-de Wijer et al. found higher DNA concentrations in buccal samples from males compared to females [11], while Verdon et al.  reported higher yields in females using a mouthwash collection method [13]. These differences may arise from factors like variations in epithelial thickness, cell turnover rates, and hormone levels between genders affecting cellular content and abundance in oral mucosa [14]. However, other studies found no significant gender difference in yields or purity [15, 16]. Due to this variability across existing literature, it is important to further assess whether intrinsic gender attributes impact susceptibility of buccal DNA to impurities during collection and extraction. Evaluating gender trends could help optimize protocols and improve standardization.

In light of these considerations, this paper endeavors to compare DNA concentrations obtained from buccal swab samples between males and females in the Volta Region of Ghana by assessing and comparing key metrics like DNA concentration, A260/280 and A260/230 ratios between males and females in this population.

All procedures and standard recommendations at the sample collection stage and the laboratory phase were followed in this study [3].

Study Population

A total of 240 buccal swab samples were collected from one main ethnic group in Ghana (Ewe) The volunteers were recruited from the general population of Ghana. This was after the School of Medicine and Dentistry and the Committee on Human Research, Publications and Ethics of Kwame Nkrumah University of Science and Technology (KNUST)- Ghana, gave ethical approval for the study to be carried out (CHRPE/AP/491/22). Related volunteers from educational institutions, and the communities aged between 18-90 years donated their buccal samples with informed consent. Samples were taken from 125 males and 115 females.

Sample Collection

Buccal swab samples were collected from volunteers aged 18-90 years who provided informed consent. To ensure cleanliness, sampling was performed in enclosed clinical environments using proper sanitary precautions. Participants were asked to rinse their mouths with sterile saline solution before sample collection to remove food debris. Sterile EUROTUBE® Collection Swabs (deltalab, Spain) were used to collect all buccal samples by swabbing the interior cheeks and gums. Swabbing was done using consistent pressure and technique for all subjects. The swabs were immediately transferred to sterile 500 μL microcentrifuge tubes prefilled with cell lysis buffer (Qiagen, UK) and sealed to avoid environmental contamination. The tubes were placed in cold storage and transported to the laboratory for processing under temperature-controlled conditions. Strict contamination controls were followed throughout sample acquisition to prevent introduction of impurities during buccal cell collection. Participants were asked to refrain from eating, drinking, smoking, or oral hygiene procedures for at least 30 minutes prior to sample collection. This fasting helps remove food residues and minimize bacterial contamination. They were then made to rinse their mouths thoroughly with 20mL sterile phosphate buffered saline for 30 seconds immediately before sampling. This helped clear the oral cavity of debris and loose epithelial cells. The participants were seated upright in a relaxed position under adequate lighting during swabbing to ensure visibility and access to the oral cavity.

Standardization of The Buccal Swab Sampling Technique

The same type of swab was used for all samples. Participants were swab with the same motion (e.g. back and forth) inside cheek for 3 to 5 strokes to collect averagely equal sample amount. After collection, immediately the swabs were stored in 1mL of lysis buffer under room temperature conditions.

Participants were made to rinse mouth with same volume of sterile water/saline for 30 seconds to one minute beforehand to remove food residues. Individuals with oral lesions, bleeding gums, recent dental work, smoking or tobacco were excluded and used to remove confounders.

Collectors were trained to perform the technique consistently with minimal variation in number of strokes, pressure, area swabbed. The same cheek area was swabbed for each participant and equal amount of lysis buffer was used to digest swabs during extraction.

By controlling as many variables as possible using standardized collection materials, oral preparation, storage, technician training, and extraction protocols, the sampling technique was optimized for consistency across all samples. This helped improve data quality and reliability. The calibration methods and purity ratio thresholds as described by Lucena-Aguilar et al., (2016) were adopted to ensure properly standardized and optimized DNA quantification in the current study

DNA Extraction

All the samples were extracted using the Puregene (Qiagen® UK) extraction method with some in-house modification to the manufactures protocol. The original protocol requires the use of 300 µL of the cell lysis buffer (Qiagen, UK) but this was modified to 600 µL for this study. The reason for the modification was to have enough buffer around the swab to prevent bacteria growth on dry areas of un-soaked swabs. The rest of the manufacturer’s protocol remained the same.

The first step in the extraction process involved the addition of 100 µL of the cell lysis buffer Qiagen UK), to the 2 mL test tube containing the 500 µL buffer (Qiagen, UK) and swabs from the field. This is to disrupt and lyse the cell membrane and plasma membrane of cells and organelles. It was vortexed vigorously for 10 seconds.  A volume of 200 µL of the buffer around the swab in the vortexed tube was aliquoted into a 1.5 mL microcentrifuge tube. 3µL of puregene proteinase K 10 mg/mL was added and mixed by inverting 10-15 times and incubated at 56 ?C for 20 minutes (the tubes were vortexed every 10 minutes) for complete cell lysis. From this sample, 100 µL of protein precipitation solution was added and vortexed vigorously at high speed for 30 seconds and incubated on ice for 5 minutes.

After the ice incubation, the test tube was centrifuged at 13000 rpm for 3 minutes and the supernatant from centrifugation was carefully decanted into a 1.5 mL volume microcentrifuge tube containing a 300 µL of pre-aliquoted isopropanol, leaving the protein pellet in the tube, the new tube was mixed by inversion. It was then centrifuged at 13000 rpm for 5 minutes, and the supernatant discarded with care, leaving the pellet inside. The tube was drained on a clean absorbent paper.

To wash the DNA pellet, 300 µL of 70% ethanol was added and centrifuged for 1 minute and the supernatant carefully discarded. The tube was air-dried for 5-10 minutes. The DNA pellet was hydrated with 50 µL DNA hydration solution and left on the bench to dissolve for about 10 minutes.

DNA Concentration Determination

Total DNA was extracted from buccal swab samples using a Puregene DNA extraction kit with modifications to the manufacturer's protocol. DNA quantitation was then performed using a Thermo Scientific™ NanoDrop™ spectrophotometer. The purity levels associated with the DNA concentrations were also determined for A260/280 with a UV absorbance ratio of 1.8 < R < 2.0 considered to be good purified DNA (van Wieren-de Wijer et al., 2009) and A260/230 with a UV absorbance ratio commonly within the range between 2.0 and 2.2 considered to be good purified DNA (Lucena-Aguilar et al., 2016). 2μL of each DNA extract was loaded onto the pedestal and measured using the DNA-50 mode to assess absorbance at 260nm. The NanoDrop software automatically calculated DNA concentration in ng/μL based on the sample absorbance, using the Beer-Lambert law (Hindash & Hindash, 2022) and an extinction coefficient of 50 ng/μL for double-stranded DNA at 260nm. Three measurements were taken per sample and averaged. The process was repeated for all 240 samples, and DNA concentrations compared between male and female groups using statistical analysis.

Statistical Analyses

The statistical software SPSS was utilized for data analysis. A two-tailed independent samples t-test was conducted to compare mean DNA concentrations between males and females. The Levene's test assessed homogeneity of variances. Mean DNA concentrations were calculated for samples with acceptable as opposed to unacceptable salt and protein purity levels based on A260/230 and A260/280 ratios respectively. An independent samples t-test evaluated whether the difference in means was statistically significant. For gender distribution of purity levels, a chi-square test was applied. All results were considered significant at p<0.05. Data is presented as mean ± standard deviation. Statistical tests determined whether DNA yields and purity showed gender dependence in the study population.

We sought to determine whether DNA concentrations obtained from buccal swabs as well as the impurities present were dependent on gender. DNA was extracted from all the buccal swabs. Although the Sterile EUROTUBE® Collection Swab (deltalab, Spain) was used in this study, it could not achieve high DNA yields. DNA yield of buccal swabs ranged from 0.8ng/uL to 111.4ng/uL. The buccal swab collection method yielded an average DNA concentration of 28.6 ng/uL. The quality of DNA can have an impact on the quantity of DNA and this was evident in the purities determined. For A260/280, the purity levels for both males and females were approximately ranging from 0.68 to 1.87 and between 0.10 to 3.04 for A260/230 both indicating unacceptable levels of protein, salt and alcohol contaminants.

Regarding the impurities, there were generally high levels of impurities determined in most samples however a majority of those samples recording high levels of impurity were low in DNA concentration (<20ng/uL). This was true for the A260:280 ratios. An A260/280 ratio much higher than the expected 1.8 indicates possible leftover RNA in the sample, which absorbs strongly at 260nm. Also, the breakdown of proteins into aromatic amino acids during extraction could increase absorbance at 280nm. Almost all samples recorded poor A260:230 ratios. A poor A260/230 ratio, as was observed in many of the buccal DNA samples in this study, indicates the presence of contaminants that absorb light at 230nm. Samples with higher DNA concentrations recorded higher levels of impurities as compared to those with lower levels of impurities. Thus, these samples recorded lower A260:230 ratios outside of the acceptable range.

The average DNA concentration in females was higher than in males. However, there was no statistically significant difference between the two groups (Table 1). The slightly higher mean concentration in females contradicts some previous studies that found higher DNA yields in males (van Wieren-de Wijer et al., 2009) who observed DNA concentration to be higher in buccal swab samples from males than from females aligns with another report of higher concentrations in female samples using a mouthwash collection method [13]. Several factors may account for the variability in DNA yields across genders. Hormonal differences can affect epithelial cell turnover rates and abundance in the buccal mucosa between males and females. While males tend to have higher epithelial proliferation, females may have thicker buccal epithelium resulting in more DNA [14]. Inconsistent buccal swabbing pressure and efficiency of cell lysis during extraction can also impact yields. Overall, while mean DNA concentrations were not significantly different, individual biological variation makes it difficult to generalize gender trends in buccal DNA content.

Table 1: Mean concentrations of variables studied.

5 samples had acceptable levels of salt impurities (2.0-2.2) while 235 samples had unacceptable levels of salt impurities. The mean DNA concentration of samples with acceptable salt impurities was lower than those with unacceptable salt impurities. The difference was however statistically insignificant (Table 1). Although very few, only males had salt purity levels within acceptable limits (Table 1). The extremely low proportion of samples with adequate salt purity corroborates the findings of poor A260/230 ratios in the study's results. The higher prevalence of unacceptable salt contamination in female samples is concerning and indicates possible issues with compliance to oral rinsing protocols prior to buccal swabbing. Compared to a study by Scott et al. (Scott, 2016) that found 7% of buccal samples had acceptable A260/230 ratios, the current study indicates even poorer salt purities overall. However, the distribution was opposite, with Scott et al. reporting 60% of samples with acceptable purities being from females. This contrast highlights the variability in contamination across studies. On the contrary, 25% of the total samples had acceptable levels of protein impurities [~1.8(1.7-2.0)] while 75% had unacceptable levels of protein impurities. Despite this variation, the mean DNA concentration of samples that had acceptable protein impurities was significantly higher than those with unacceptable protein impurities (Table 1). The results differ from a study by Lucena-Aguilar et al., (2016) that found no significant correlation between DNA yield and protein impurities, yet they align with findings of the same study as it reported lower DNA concentrations in buccal samples with A260/280 ratios outside the desired 1.7-2.0 range. The reduced yields in samples with unacceptable protein contamination could be due to the inhibitory effects of proteins on DNA extraction and enzymatic processing. Proteases can degrade DNA, while denatured proteins can bind to DNA and interfere with quantification (Reinking et al., 2020). While purified genomic DNA ideally should have A260/280 ratios close to 1.8, the current data reinforces that even small deviations negatively impact yields. Tight control over pre-extraction steps like cell lysis and removal of non-DNA organic material is necessary to minimize protein contamination. The findings highlight the importance of sample purity for maximizing DNA recovery from buccal swabs.

A higher proportion of male samples had acceptable protein ratios within 1.7-2.0 compared to female samples (Table 1) indicating lower susceptibility of male buccal swabs to protein contamination during DNA extraction. A potential reason could be that the thicker epithelial lining and higher cell content in female buccal mucosa releases more cellular proteins that persist through extraction. Males also tend to have higher rates of epithelial turnover that may reduce protein buildup. However, other studies like Nauwelaerts et al., and Nishita et al., found no significant gender difference in buccal DNA protein impurities [15,16]. Variability in collection methods, extraction protocols, and demographic factors across studies make trends difficult to generalize. The inconsistency in contamination level observed in this study could be as a result of variance in sampling techniques as the amount of force exerted during sample collection by the technician varies from one participant to the other in a bid to prevent and in the worst case reduce bleeding.

While this data implies males had better protein purities, the small sample size of acceptable purity samples warrants caution when interpreting gender patterns. Larger balanced groups and standardized procedures could better elucidate if gender intrinsically affects propensity for protein contamination in buccal DNA samples.

The idea of using buccal cells for DNA collection was presented as a workable and affordable strategy. This claim was made in light of the fact that buccal cells can be easily collected and transported by mail using FTA cards while being non-invasive. However, it is essential to make sure that the mouth is thoroughly rinsed before collecting buccal cells. Lack of proper mouth-washing increases the risk of contamination, which can result in the presence of contaminants [10]. Certain food contaminants absorb light at 230 nm, which is the wavelength used to assess the presence of contaminants such as salt and protein in the samples. For example, some food seasonings, or spices may contain compounds that have absorbance peaks at 230 nm. When these impurities are present in the DNA sample, they can increase the absorbance at 230 nm, leading to a lower A260/230 ratio [17].

Several factors can account for the low yield of DNA in this study. This notwithstanding, for applications like PCR amplification, maintaining the purity of extracted DNA is more crucial than increasing its yield. The quality of DNA samples can be impacted by numerous circumstances. Possible pollutants include contaminants including polysaccharides, humic acid (found in food), milk proteins, lipids, and phenolic compounds. These impurities can decrease DNA purity, denature enzymes, cause DNA degradation, and have a detrimental impact on DNA solubility. Even compounds like EDTA, phenolic compounds, and NaOH that are contained in the reagents used for DNA extraction can have negative effects. Additionally, isopropanol and ethanol can drastically reduce DNA purity when used in the DNA precipitation and wash operations. It is critical to keep the purity high and reduce the presence of these impurities to enable reliable downstream applications [18].

Although not investigated in this current study, dietary and lifestyle factors have also been shown to have genotoxic effects on the oral cavity and have the ability to damage DNA [19]. Participants were provided with commercially produced water to rinse their mouths before sampling was done. However, this was not strictly monitored by the researchers. This could be another reason for the low DNA yield observed in this study.

This study found that DNA extracted from buccal swabs can contain high levels of contaminants that compromise purity. Though buccal swabbing is a simple DNA collection method, the quality of extracted DNA is vulnerable to impurities.

A key finding was the high prevalence of poor A260/230 ratios, especially in female samples, indicating the presence of contaminants like salts, organic compounds, and solvents. Inadequate oral rinsing prior to sampling likely retained impurities from food, drinks, or smoking. The DNA extraction method may have also introduced contaminants from reagents.

While mean DNA yields did not significantly differ between males and females, concentrations were generally low. Lifestyle factors altering oral microbiota and inadequate mouth washing may lower cell yields from buccal swabbing.

To improve purity, strict protocols are needed for thorough oral rinsing before buccal swab collection. Monitoring compliance can reduce retained contaminants. Optimizing extraction methods by removing problematic reagents or adding steps to remove impurities could also enhance DNA quality.

Though buccal swabbing has advantages for easy non-invasive sampling, controlling contamination is critical to obtain DNA of sufficient purity for sensitive downstream applications like PCR and sequencing. With improved protocols to reduce impurities, buccal swabs can remain a valuable DNA source for genetics research.

Authors’ Contributions

HO carried out sampling and wrote the first draft of the manuscript. SAK and EUO participated in the conceptualization and the design of the research protocol and wrote the final manuscript. All authors participated in the laboratory analysis as well as read and approved the final draft of the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics Approval

The Committee on Human Research, Publications and Ethics of Kwame Nkrumah University of Science and Technology (KNUST)- Ghana, gave ethical approval with reference number (CHRPE/AP/491/22) for the study to be carried out.

Acknowledgment

The authors of this work are grateful to the entire participants that availed themselves for this study

Data Availability Statement

All data used for the study are available upon a reasonable request.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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