Oragene discover kit

Manufactured by DNA Genotek

Sourced in Canada

The Oragene Discover kit is a non-invasive DNA collection device that allows for the collection and stabilization of saliva samples. The kit contains materials necessary for the collection, preservation, and transportation of DNA samples.

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Lab products found in correlation

Qubit fluorometer, by Thermo Fisher Scientific (2 mentions) Taqman universal master mix, by Thermo Fisher Scientific (1 mentions) Cfx connect, by Bio-Rad (1 mentions) Qiaamp dna kit, by Qiagen (1 mentions) Pyromark md system, by Qiagen (1 mentions) Pyromark pcr kit, by Qiagen (1 mentions) Gentra puregene kit, by Qiagen (1 mentions) 25 1506 1pf tt mc, by Puritan (1 mentions) Puregene dna purification kit, by Qiagen (1 mentions) Ez dna methylation kit, by Zymo Research (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Amplitaq gold 360 master mix, by Thermo Fisher Scientific (1 mentions) 9700 thermocycler, by Thermo Fisher Scientific (1 mentions)

12 protocols using oragene discover kit

FKBP5 SNP Genotyping in Children

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Saliva samples were obtained using the Oragene DISCOVER kits (OGR-575) for Assisted Collections (DNA Genotek, Kanata, Ontario, Canada), and DNA was isolated following the manufacturer’s instructions. DNA samples were genotyped for the FKBP5 SNP rs1360780 through an allelic discrimination assay using predesigned Taqman primers (part #C_8852038_10, Life Technologies) and Taqman universal master mix (Life Technologies) via established protocols as directed by the manufacturer on a Bio-Rad CFX connect. One hundred and twelve children were homozygotes for the C allele, 100 children were CT heterozygotes, and 19 children were homozygotes for the T allele. Longitudinal models for hypothesis testing utilized a dichotomous variable that included children with the CC genotype versus children with the T risk allele (heterozygous or homozygous).

Parade S.H., Parent J., Rabemananjara K., Seifer R., Marsit C.J., Yang B.Z., Zhang H, & Tyrka A.R. (2017). Change in FK506 binding protein 5 (FKBP5) methylation over time among preschoolers with adversity. Development and psychopathology, 29(5), 1627-1634.

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Saliva DNA Extraction and Quantification

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Saliva was collected using ORAgene® DISCOVER kits (OGR-500, DNA Genotek Inc., Ottawa, ON, Canada) and processed per the manufacturer’s protocol. Genomic DNA was extracted using a QIAamp DNA Kit (Qiagen Inc., Germantown, MD, United States). DNA was quantified using a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, United States).

Wen Y.F., Gaedigk A., Boone E.C., Wang W.Y, & Straka R.J. (2022). The Identification of Novel CYP2D6 Variants in US Hmong: Results From Genome Sequencing and Clinical Genotyping. Frontiers in Pharmacology, 13, 867331.

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Methylation Analysis of HTR2A Promoter

Cited 1 time

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Two CpGs (−1420 and −1224) were studied based on findings of Falkenberg and colleagues (2011) (link) and Paquette and colleagues (2013) (link). Saliva samples were obtained using the Oragene DISCOVER kits (OGR-575) for Assisted Collections (DNA Genotek, Kanata, Ontario, Canada), and DNA was isolated following the manufacturer’s instructions. A 276 bp. region of the HTR2A promoter region (Chr 7: 28415063-28414801) was amplified from bisulfite modified DNA using the pyromark PCR kit (Qiagen, 203443), and DNA methylation assessed through pyrosequencing following a serial pyrosequencing protocol using a PyroMark MD system (Qiagen) and two sequencing primers as described in Paquette et al (2013) (link). Each pyrosequencing assay contained a bisulfite conversion control to assess conversion efficiency. All samples examined demonstrated >95% conversion.

Parade S.H., Novick A.M., Parent J., Seifer R., Klaver S.J., Marsit C.J., Gobin A.P., Yang B.Z, & Tyrka A.R. (2017). Stress exposure and psychopathology alter methylation of the serotonin receptor 2A (HTR2A) gene in preschoolers. Development and psychopathology, 29(5), 1619-1626.

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Genetic Evaluation of XDP Patients

Cited 2 times

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Subjects recruited for this study included individuals with XDP evaluated at Massachusetts General Hospital (Boston), Jose R. Reyes Memorial Medical Center (Manila, Philippines), and regional clinics on the island of Panay (Philippines). All participants provided written informed consent, and the study was approved by all institutional review boards. Clinical evaluation included comprehensive neurological examinations with recorded scores for standard scales: Burke–Fahn–Marsden, Tsui–Torticollis, Toronto Western Spasmodic Torticollis Rating, and Voice Disability Index (91 (link)). Blood was collected from all patients, and a subset also provided saliva specimens. gDNA was extracted from blood using the Gentra Puregene kit (Qiagen) and from saliva using the Oragene Discover kit (DNA Genotek). Enrolled subjects were confirmed to be positive for six of the seven known haplotype markers (five DSCs and the SVA) by PCR amplification of blood gDNA followed by Sanger sequencing of amplicons as previously described (55 (link)). The collection methods and the clinical characterization of donor subjects who provided archival DNA specimens included in this study have been previously described (20 (link)).

Bragg D.C., Mangkalaphiban K., Vaine C.A., Kulkarni N.J., Shin D., Yadav R., Dhakal J., Ton M.L., Cheng A., Russo C.T., Ang M., Acuña P., Go C., Franceour T.N., Multhaupt-Buell T., Ito N., Müller U., Hendriks W.T., Breakefield X.O., Sharma N, & Ozelius L.J. (2017). Disease onset in X-linked dystonia-parkinsonism correlates with expansion of a hexameric repeat within an SVA retrotransposon in TAF1. Proceedings of the National Academy of Sciences of the United States of America, 114(51), E11020-E11028.

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Comprehensive Epilepsy Tissue Profiling

Cited 1 time

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A total of 77 brain tissue samples from 40 subjects were obtained after clinically necessary resections as determined by electrode-guided epileptic focal points. In patients with more than one area resected, a single sample was selected to be consistent in processing among subjects. Sample selection was informed by the likelihood of the tissue type based on the database of methylation profiles (https://www.molecularneuropathology.org/mnp) (Capper et al., 2018 (link)). Exclusion criteria of samples included <30% consistency in methylation profile of samples, profiles suggestive of malignancy (n = 3), or inflammatory tumor microenvironment (n = 1). Subsequently, in total, 36 brain samples from 36 subjects were used for downstream analysis. Samples were labeled with the corresponding brain regions and immediately transported on dry ice and stored at −80 °C until downstream DNA extraction and DNAm analysis.
Whole blood samples were collected in EDTA tubes in the surgical suite after the completion of the procedure, but prior to wakening the patient. In total, 37 whole blood samples were further analyzed for the current study. 23 saliva were obtained after surgery via the Oragene DISCOVER^™ kit (DNA Genotek Inc., OGR-500) along with 26 buccal tissue collected using swabs (Puritan, 25–1506 1 PF TT MC), which were obtained within 2 days post-surgical intervention.

Takiff H.E., Cimino M., Musso M.C., Weisbrod T., Martinez R., Delgado M.B., Salazar L., Bloom B.R, & Jacobs WR J.r. (1996). Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis.. Proceedings of the National Academy of Sciences of the United States of America, 93(1), 362-366.

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DNA Isolation from Blood and Saliva

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DNA was isolated from four subjects from 10 ml of peripheral blood leukocytes using a DNA isolation kit (PureGene, Qiagen, Valencia, CA) according to the manufacturer’s protocol. DNA was also isolated from saliva (n = 5) using a kit (Oragene-DISCOVER kit, DNA Genotek Inc., Ottawa, Canada) according to the manufacturer’s protocol. The present genotyping procedures were found to perform consistently for DNA collection methods (blood and saliva), and for a subset of subjects both were used. All comparisons of the genotype results were confirmed across specimens.

Atilano S.R., Kenney M.C., Briscoe A.D, & Jameson K.A. (2020). A two-step method for identifying photopigment opsin and rhodopsin gene sequences underlying human color vision phenotypes. Molecular Vision, 26, 158-172.

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Genomic DNA Extraction from Saliva and Buccal Cells

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Biological samples were collected from children and their families to obtain genomic DNA. For saliva collection, a minimum of 2 mL of saliva was collected from children using the Oragene Discover kit (DNA Genotek Inc., Canada). Buccal cell sampling utilizing Isohelix DNA buccal swabs (SK-2 swabs; Cell Projects Ltd., UK) was the alternative sampling method when there was no cooperation for saliva collection. These kits provided a safe and reliable method of obtaining genomic DNA for future sequencing analysis, allowing sample storage and transportation to the research lab at room temperature. Genomic DNA was subsequently extracted through an established protocol using the Puregene DNA Purification kit (Qiagen, USA) for saliva and the Isohelix DNA Isolation kit for buccal cell samples.

Fonteles C.S., Finnell R.H., Lei Y., Zurita-Jimenez M.E., Monteiro A.J., George T.M, & Harshbarger R.J. (2021). De novo ALX4 variant detected in child with non-syndromic craniosynostosis. Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas, 54(11).

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Bisulfite Conversion and Pyrosequencing for DNA Methylation Analysis

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From each child, a saliva specimen was collected during the home visit using the Oragene-DISCOVER kit (OGR-500, DNA Genotek, Inc, Ontario, Canada). DNA bisulfite conversion was accomplished using EZ DNA Methylation Kits (Zymo Research) by following the manufacturer’s manual. Briefly, 0.5–1.0 μg of genomic DNA was first mixed with 5 μl of M-Dilution Buffer and incubated at 37 °C for 15 min and then mixed with 100 μl of CT Conversion Reagent. Mixtures were incubated in a thermocycler with 16 thermal cycles at 95 °C for 30 s and 50 °C for 1 h. Bisulfite-converted DNA samples were loaded onto 96-column plates provided in the kit for desulfonation and purification. The concentration of eluted DNA was measured using a NanoDrop 1000 spectrometer.
DNA methylation was quantitated using bisulfite-PCR and pyrosequencing using the following kits from Qiagen: ICAM1_02 for ICAM-1, CRH_01 for CRH, LEP_01 for LEP, and NOS3_02 for LINE-1. Sequencing was conducted using Pyromark Q24, and percent methylation was computed using the Pyromark Q24 Software v.2 in CpG mode. Details about the location of the measured CpG sites are in the online supporting information (Additional file 1: Table S1).

Dunstan J., Bressler J.P., Moran T.H., Pollak J.S., Hirsch A.G., Bailey-Davis L., Glass T.A, & Schwartz B.S. (2017). Associations of LEP, CRH, ICAM-1, and LINE-1 methylation, measured in saliva, with waist circumference, body mass index, and percent body fat in mid-childhood. Clinical Epigenetics, 9, 29.

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Saliva DNA Extraction and Purification

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Saliva DNA was collected and purified using the Oragene-Discover kit (DNA Genotek) following the manufacturer instructions. Saliva DNA was extracted using DNeasy Blood and Tissue kit (Qiagen) with the following modifications: 5 ml AL-buffer and 200 μl Proteinase K were added to saliva and incubated at 5600B0030C for 30 minutes. RNA was digested using 2003B0043l RNAse A (Qiagen) for 5 minutes and DNA was extracted using 4 extraction columns in parallel to optimize the yield.

Sarangi V., Jang Y., Suvakov M., Bae T., Fasching L., Sekar S., Tomasini L., Mariani J., Vaccarino F.M, & Abyzov A. (2022). All2: A tool for selecting mosaic mutations from comprehensive multi-cell comparisons. PLoS Computational Biology, 18(4), e1009487.

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Triallelic VNTR Genotyping Protocol

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Saliva samples were collected using an Oragene-DISCOVER Kit (DNA Genotek, Canada) from all subjects after they finished their pretest. Genomic DNA was extracted using the standard method supplied by the same kit. The polymerase chain reaction (PCR) for the triallelic VNTR was performed using a 9700 thermocycler (Applied Biosystems, USA) in 10 μl amplification mixture containing 0.5 mm of each primer (forward: 5′-CGGAGCAAGCCTGCCAG-3′ and backward: 5′-TGAGGGGACGACAAAGG-3′), 1× AmpliTaq Gold™ 360 Master Mix (Applied Biosystems, USA), and 30 ng template DNA. After an initial 5 min at 96 °C, the PCR proceeded with 36 cycles of 96 °C for 30 s, 51 °C for 30 s, and 72 °C for 30 s. After a final 10 min at 72 °C, the PCR was terminated at 4 °C. The PCR products for two, three, and four repeats were 240 bp, 276 bp, and 312 bp, respectively. They were separated by electrophoresis in 3% agarose gels. Each sample was genotyped twice. Genotypes were read by two researchers. Of the total sample, we observed only three samples with the four-repeat allele. We excluded them from further data analysis due to the small sample size.

Zhao W., Zhang Q., Chen X., Li Y., Li X., Du B., Deng X., Ji F., Wang C., Xiang Y.T., Dong Q., Chen C, & Li J. (2020). The VNTR of the AS3MT gene is associated with brain activations during a memory span task and their training-induced plasticity. Psychological Medicine, 51(11), 1927-1932.

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