Comparison of different collection procedures and two methods for DNA isolation from saliva (original) (raw)
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American Journal of Human Biology, 2007
Epidemiological studies may require noninvasive methods for off-site DNA collection. We compared the DNA yield and quality obtained using a whole-saliva collection device (Oragene TM DNA collection kit) to those from three established noninvasive methods (cytobrush, foam swab, and oral rinse). Each method was tested on 17 adult volunteers from our center, using a random crossover collection design and analyzed using repeated-measures statistics. DNA yield and quality were assessed via gel electrophoresis, spectophotometry, and polymerase chain reaction (PCR) amplification rate. The whole-saliva method provided a significantly greater DNA yield (mean 6 SD ¼ 154.9 6 103.05 mg, median ¼ 181.88) than the other methods (oral rinse ¼ 54.74 6 41.72 mg, 36.56; swab ¼ 11.44 6 7.39 mg, 10.72; cytobrush ¼ 12.66 6 6.19, 13.22 mg) (all pairwise P < 0.05). Oral-rinse and whole-saliva samples provided the best DNA quality, whereas cytobrush and swab samples provided poorer quality DNA, as shown by lower OD 260 /OD 280 and OD 260 /OD 230 ratios. We conclude that both a 10-ml oral-rinse sample and 2-ml whole-saliva sample provide sufficient DNA quantity and better quality DNA for genetic epidemiological studies than do the commonly used buccal swab and brush techniques. Am.
Scientific reports, 2017
Saliva has attracted attention as a diagnostic fluid due to the association of oral microbiota with systemic diseases. However, the lack of standardised methods for saliva collection has led to the slow uptake of saliva in microbiome research. The aim of this study was to systematically evaluate the potential effects on salivary microbiome profiles using different methods of saliva collection, storage and gDNA extraction. Three types of saliva fractions were collected from healthy individuals with or without the gDNA stabilising buffer. Subsequently, three types of gDNA extraction methods were evaluated to determine the gDNA extraction efficiencies from saliva samples. The purity of total bacterial gDNA was evaluated using the ratio of human β-globin to bacterial 16S rRNA PCR while 16S rRNA gene amplicon sequencing was carried out to identify the bacterial profiles present in these samples. The quantity and quality of extracted gDNA were similar among all three gDNA extraction metho...
Rev. odonto ciênc. (Online), 2010
Purpose: This study evaluated the quality of DNA obtained from stored human saliva and its applicability to human identification. Methods: The saliva samples of 20 subjects, collected in the form of saliva in natura and from mouth swabs and stored at-20ºC, were analyzed. After 7 days, the DNA was extracted from the 40 saliva samples and subjected to PCR and electrophoresis. After 180 days, the technique was repeated with the 20 swab samples. Results: The first-stage results indicated that DNA was successfully extracted in 97.5% of reactions, 95% of saliva in natura and 100% of swab saliva samples, with no statistically significant difference between the forms of saliva. In the second phase, the result was positive for all 20 analyzed samples (100%). Subsequently, in order to analyze the quality of the DNA obtained from human saliva, the SIX3-2 gene was tested on the 20 mouth swab samples, and the PCR products were digested using the MbO1 restriction enzyme to evaluate polymorphisms in the ADRA-2 gene, with positive results for most samples. Conclusion: It was concluded that the quantity and quality of DNA from saliva and the techniques employed are adequate for forensic analysis of DNA.
Background: Obtaining high quality genomic DNA safely and economically is vital for diverse studies of large populations aimed at evaluating the role of genetic factors in susceptibility to disease. Aim: This study was to test a protocol for the extraction of high quality genomic DNA from saliva samples obtained with mouthwash and taken from patients with periodontal disease. Methods: Saliva samples were taken from 60 patients and then stored at room temperature. DNA extraction was carried out at distinct post-sampling times (10, 20 and 30 days). Evaluation of genomic DNA was performed with spectrophotometry, electrophoresis, and PCR genotyping and sequencing. Results: The greatest concentration of DNA obtained was 352 mg at 10 days post-sampling, followed by 121.025 mg and 19.59 mg at 20 and 30 days, respectively. When determining the purity of DNA with the spectrophotometric ratio of 260/230, the relations of 1.20, 1.40 and 0.781 were obtained for 10, 20 and 30 days, respectively. In all samples, it was possible to amplify the product of 485 bp and the sequence of the amplicons showed 95% similarity to the reference sequence. Conclusion: The present protocol represents
Saliva as a source of genomic DNA for genetic studies: review of current methods and applications
Oral health and dental management, 2014
DNA isolated from saliva has become an attractive alternative to the use of blood-derived DNA in genetic studies and is now extensively used in many applications. This review addresses advantages of saliva DNA for large population-based studies and points out caveats for use in certain techniques. We compiled data comparing saliva-derived genomic DNA to blood-derived DNA with regards to quality, quantity and convenience. Special attention is given to the usefulness of saliva-derived DNA for PCR-based methods and genome-wide analyses.
Arquivos em Odontologia, 2023
Aim: This study aimed to verify the quantity and purity of DNA obtained from buccal cells under different storage conditions. Methods: Thirty students, between 18 and 23 years of age participated in the study. Three samples of genetic material were collected from each student (samples A, B, and C) through a mouth rinse with 5 mL of 3% glucose. The first phase of DNA extraction from sample A was carried out on the same day of sample collection, whereas samples B and C were stored in a refrigerator and a freezer, respectively, for 1 month before the first extraction phase. DNA extraction was performed with 10 M ammonium acetate and 1 mM EDTA. Sample purity was assessed by spectrophotometry. Statistical analyses were performed through descriptive analysis and analysis of variance ANOVA using the SPSS software, version 21.0. Results: the samples presented no statistically significant differences between the DNA quantity (p = 0.37) or quality (p = 0.16). Conclusion: the quantity and purity of DNA extraction from the three samples were satisfactory, and no differences in storage conditions were found. Uniterms: DNA. Mouth. Mucosa.
Sift Desk Journals, 2018
INTRODUCTION Genomic DNA has become a very important source for multiple purposes in research projects. Therefore, the analysis of the quality of the extraction of DNA using different specimen collection methods; are part of every materials and method section in current papers (1-7). The objective of this study was to evaluate which protocol extracts the highest yield with better quality of DNA from human buccal swabs. There are two different ways to harvest DNA from buccal swabs: single or multi-step procedure. Our study compared the quality of the DNA extracted using these two different methods of DNA extraction; though the determination spec-troscopic measurements at 280, 260, and 230 nm wavelengths and the suitability for application in geno-typing assays.
evaluation of oral Cavity DNA extraction Methods on Bacterial and Fungal Microbiota
Scientific Reports, 2019
The objective of this study was to evaluate the most effective method of DNA extraction of oral mouthwash samples for use in microbiome studies that utilize next generation sequencing (NGs). eight enzymatic and mechanical DNA extraction methods were tested. Extracted DNA was amplified using barcoded primers targeting the V6 variable region of the bacterial 16S rRNA gene and the ITS1 region of the fungal ribosomal gene cluster and sequenced using the Illumina NGS platform. Sequenced reads were analyzed using QIIME and R. The eight methods yielded significantly different quantities of DNA (p < 0.001), with the phenol-chloroform extraction method producing the highest total yield. There were no significant differences in observed bacterial or fungal Shannon diversity (p = 0.64, p = 0.93 respectively) by extraction method. Bray-Curtis beta-diversity did not demonstrate statistically significant differences between the eight extraction methods based on bacterial (R 2 = 0.086, p = 1.00) and fungal (R 2 = 0.039, p = 1.00) assays. No differences were seen between methods with or without bead-beating. These data indicate that choice of DNA extraction method affect total DNA recovery without significantly affecting the observed microbiome.
Methods and Protocols
(1) Introduction: Due to the non-invasive nature of saliva, many methods have been used to isolate and collect DNA from saliva samples for microbial screening. Many oral microbes also inhabit the oral biofilm, which may represent significantly different microbial constituents that may contribute to oral health and disease, including caries and periodontal disorders. Moreover, the biofilm may vary within the same patient at different sites. Few studies have evaluated the comparison between DNA isolated from saliva and DNA from site-specific biofilm, with virtually no studies addressing this analysis among pediatric patients. (2) Methods: An existing repository of paper point derived biofilm, gingival crevicular fluid (GCF), and unstimulated saliva samples previously collected from pediatric patients (n = 47) was identified. DNA was isolated from biofilm sites (tongue, upper buccal molar, mandibular lingual incisor), and GCF and saliva were used for quantitative DNA comparison using a phenol:chloroform extraction. A quantitative and qualitative analysis was performed using the NanoDrop 2000 spectrophotometer using absorbance readings at A230 nm, A260 nm and A280 nm. (3) Results: These data demonstrated the successful isolation of DNA from all of the patient samples, with the highest concentrations observed among unstimulated saliva (4264.1 ng/µL) and the lowest derived from GCF (1771.5 ng/µL). No differences were observed between males and females or minorities and non-minority patients. In addition, comparison of the overall concentrations of DNA obtained from adult samples was slightly higher than, but not significantly different from, the concentrations obtained from pediatric samples (p = 0.2827). A real-time quantitative qPCR screening revealed that all of the samples evaluated harbored bacterial and human DNA of sufficient quantity and quality for a molecular screening greater than the limit of detection (∆Rn = 0.01). (4) Conclusions: Many methods are currently available to provide the sampling and screening of saliva and specific sites within the oral cavity, but the validation and comparison of simple and low-cost methods, that include paper point sampling and unstimulated saliva collection, may suggest these methods and protocols provide sufficient DNA quality and quantity for molecular screening and other comparison applications. In addition, although heterogeneity will be a constant and consistent feature between patient samples, standardized methods that provide similar and consistent DNA from various oral sites may provide needed consistency for screening and molecular analysis.