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Design of a protocol for obtaining genomic DNA from saliva using mouthwash: Samples taken from patients with periodontal disease.

ABSTRACT: 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 ?g at 10 days post-sampling, followed by 121.025 ?g and 19.59 ?g 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 an easy, safe and economical technique for obtaining high quality genomic DNA.

SUBMITTER: Mendoza AC

PROVIDER: S-EPMC4862237 | biostudies-literature | 2016 May-Aug

REPOSITORIES: biostudies-literature

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Design of a protocol for obtaining genomic DNA from saliva using mouthwash: Samples taken from patients with periodontal disease.

Mendoza Ángel Chávez ÁC Volante Beatriz Buentello BB Hernández María Esther Ocharán ME Mendoza Claudia Camelia Calzada CC Pliego Arturo Flores AF Baptista Gonzalez Héctor A HA Juárez Higinio Estrada HE

Journal of oral biology and craniofacial research 20160211 2

<h4>Background</h4>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.<h4>Aim</h4>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.<h4>Methods</h4>Saliva samples were taken from 60 patients and then stored at room temperature. DNA extractio ...[more]

PMID: 27195211

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Project description:BackgroundTechnical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. However, usability of previously collected samples and the effects of environmental conditions on the samples during collection have not been assessed in detail. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva.MethodsSaliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP) in the catechol-0-methyltransferase gene (COMT rs4680) and one representative variable number of tandem repeats (VNTR) in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region) were selected for genetic analyses.ResultsThe smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 μg DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days), repeated freeze-thaw cycles (up to 6 cycles), and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible.ConclusionsWhole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using 10 ng of DNA per genotyping reaction, the obtained samples can be used for more than one hundred candidate gene assays. When saliva is collected with an absorbent device, most of the nucleic acid content remains in the device, therefore it is advisable to collect the device separately for later genetic analyses.

Project description:BACKGROUND: The increasing trend for incorporation of biological sample collection within clinical trials requires sample collection procedures which are convenient and acceptable for both patients and clinicians. This study investigated the feasibility of using saliva-extracted DNA in comparison to blood-derived DNA, across two genotyping platforms: Applied Biosystems Taqman™ and Illumina Beadchip™ genome-wide arrays. METHOD: Patients were recruited from the Pharmacogenetics of Breast Cancer Chemotherapy (PGSNPS) study. Paired blood and saliva samples were collected from 79 study participants. The Oragene DNA Self-Collection kit (DNAgenotek®) was used to collect and extract DNA from saliva. DNA from EDTA blood samples (median volume 8 ml) was extracted by Gen-Probe, Livingstone, UK. DNA yields, standard measures of DNA quality, genotype call rates and genotype concordance between paired, duplicated samples were assessed. RESULTS: Total DNA yields were lower from saliva (mean 24 ?g, range 0.2-52 ?g) than from blood (mean 210 ?g, range 58-577 ?g) and a 2-fold difference remained after adjusting for the volume of biological material collected. Protein contamination and DNA fragmentation measures were greater in saliva DNA. 78/79 saliva samples yielded sufficient DNA for use on Illumina Beadchip arrays and using Taqman assays. Four samples were randomly selected for genotyping in duplicate on the Illumina Beadchip arrays. All samples were genotyped using Taqman assays. DNA quality, as assessed by genotype call rates and genotype concordance between matched pairs of DNA was high (>97%) for each measure in both blood and saliva-derived DNA. CONCLUSION: We conclude that DNA from saliva and blood samples is comparable when genotyping using either Taqman assays or genome-wide chip arrays. Saliva sampling has the potential to increase participant recruitment within clinical trials, as well as reducing the resources and organisation required for multicentre sample collection.

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Design of a protocol for obtaining genomic DNA from saliva using mouthwash: Samples taken from patients with periodontal disease.

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Design of a protocol for obtaining genomic DNA from saliva using mouthwash: Samples taken from patients with periodontal disease.

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