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Saliva as an alternative source of high yield canine genomic DNA for genotyping studies.

ABSTRACT: BACKGROUND: The domestic dog presents an attractive model system for the study of the genetic basis of disease. The development of resources such as the canine genome sequence and SNP genotyping platforms has allowed for the implementation of canine genetic studies. Successful implementation of such studies depends not only on the quality of individual DNA samples, but also on the number of samples obtained. The latter can be maximized using a non-invasive DNA collection method that can increase study participation. We compared the DNA yield and quality obtained from blood and buccal swabs to those obtained using a non-invasive saliva collection kit (Oragene *ANIMAL kit). We also assessed the success rate of PCR amplification and genotyping accuracy of DNA isolated using these collection methods. FINDINGS: Comparison of DNA yields from matched saliva, blood and buccal swab samples showed that yields from saliva were significantly higher than those from blood (p = 0.0198) or buccal swabs (p = 0.0008). Electrophoretic analysis revealed that blood and saliva produced higher quality DNA than buccal swabs. In addition, a 1.1-kb PCR fragment was successfully amplified using the paired DNA samples and genotyping by PCR-RFLP yielded identical results. CONCLUSION: We demonstrate that DNA yields from canine saliva are higher than those from blood or buccal swabs. The quality of DNA extracted from saliva is sufficient for successful amplification of a 1.1-kb fragment and for accurate SNP genotyping by PCR-RFLP. We conclude that saliva presents a non-invasive alternative source of high quantities of canine genomic DNA suitable for genotyping studies.

SUBMITTER: Mitsouras K

PROVIDER: S-EPMC2781016 | biostudies-other | 2009

REPOSITORIES: biostudies-other

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Saliva as an alternative source of high yield canine genomic DNA for genotyping studies.

Mitsouras Katherine K Faulhaber Erica A EA

BMC research notes 20091029

<h4>Background</h4>The domestic dog presents an attractive model system for the study of the genetic basis of disease. The development of resources such as the canine genome sequence and SNP genotyping platforms has allowed for the implementation of canine genetic studies. Successful implementation of such studies depends not only on the quality of individual DNA samples, but also on the number of samples obtained. The latter can be maximized using a non-invasive DNA collection method that can i ...[more]

PMID: 19874604

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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.

Project description:BackgroundThe question of whether DNA obtained from saliva is an acceptable alternative to DNA from blood is a topic of considerable interest for large genetics studies. We compared the yields, quality and performance of DNAs from saliva and blood from a mostly elderly study population.MethodsTwo thousand nine hundred ten DNAs from primarily elderly subjects (mean age ± standard deviation (SD): 65 ± 12 years), collected for the Primary Open-Angle African-American Glaucoma Genetics (POAAGG) study, were evaluated by fluorometry and/or spectroscopy. These included 566 DNAs from blood and 2344 from saliva. Subsets of these were evaluated by Sanger sequencing (n = 1555), and by microarray SNP genotyping (n = 94) on an Illumina OmniExpress bead chip platform.ResultsThe mean age of subjects was 65, and 68 % were female in both the blood and saliva groups. The mean ± SD of DNA yield per ml of requested specimen was significantly higher for saliva (17.6 ± 17.8 μg/ml) than blood (13.2 ± 8.5 μg/ml), but the mean ± SD of total DNA yield obtained per saliva specimen (35 ± 36 μg from 2 ml maximum specimen volume) was approximately three-fold lower than from blood (106 ± 68 μg from 8 ml maximum specimen volume). The average genotyping call rates were >99 % for 43 of 44 saliva DNAs and >99 % for 50 of 50 for blood DNAs. For 22 of 23 paired blood and saliva samples from the same individuals, the average genotyping concordance rate was 99.996 %. High quality PCR Sanger sequencing was obtained from ≥ 98 % of blood (n = 297) and saliva (n = 1258) DNAs. DNA concentrations ≥10 ng/μl, corresponding to total yields ≥ 2 μg, were obtained for 94 % of the saliva specimens (n = 2344).ConclusionsIn spite of inferior purity, the performance of saliva DNAs for microarray genotyping was excellent. Our results agree with other studies concluding that saliva collection is a viable alternative to blood. The potential to boost study enrollments and reduce subject discomfort is not necessarily offset by a reduction in genotyping efficiency. Saliva DNAs performed comparably to blood DNAs for PCR Sanger sequencing.

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Saliva as an alternative source of high yield canine genomic DNA for genotyping studies.

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Saliva as an alternative source of high yield canine genomic DNA for genotyping studies.

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