Navigation Links
Oragene and DNA sequencing with ABI PRISM

J. Chartier
DNA Genotek Inc., Ottawa, ON, Canada

DNA from Oragene is reliably sequenced using the ABI PRISM 377 DNA Sequencer.


Introduction
Blood leukocytes are the traditional source of DNA for molecular testing, but collection is invasive and specimens require infectious precautions. Buccal swabs are a less-invasive method of collecting DNA for sequencing applications such as HLA typing and prelingual deafness screening (ref. 1, 2) but they tend to have an appreciable failure rate (ref. 3). Oragene is a non-invasive DNA self-collection kit from saliva that provides significantly higher DNA yields than buccal swabs (ref. 4). The purpose of this study was to investigate the suitability of DNA from Oragene for DNA sequencing.


Materials and Methods
DNA collection
Saliva samples were collected from 5 donors. Collection and purification of DNA was carried out according to protocols supplied with the Oragene kit.

Primer design
PCR primers for the human Thymidylate Synthase (TYMS) gene were designed based on the publicly available DNA sequence (GenBank accession no. AP001178). The primers generate a 560-bp fragment. Table 1 shows the primer sequences.

DNA Sequencing
Oragene-purified DNA from each of the 5 samples was used as the template for PCR with the TS 560 primers. The PCR products were sequenced in both directions by Cortec DNA Service Laboratories (Kingston, ON) using an ABI PRISM 377 DNA Sequencer (Applied Biosystems) and the DYEnamic ET Dye Terminator Kit (Amersham Biosciences). Prior to sequencing, the PCR products were purified using the microCLEAN DNA clean-up reagent (Microzone).


Results
DNA sequencing results were aligned using CLUSTAL W (Version 1.83), a multiple sequence alignment program. All 5 samples correctly aligned to the original human Thymidylate Synthase gene sequence. Figure 1 shows a representative ABI PRISM sequencing read-out.


Discussion and Conclusions
Oragene is a non-invasive and reliable method of collecting DNA from saliva. DNA from Oragene works well for DNA sequencing with the ABI PRISM 377 DNA Sequencer.


References
1. Liu, C., Chu, C., Lee, Y., Shi, Y., and M. Lin. (2004). Identification of two novel HLA-DRB1 alleles, DRB1*0903 and DRB1*1145. Tissue Antigens. 64: 99101.

2. Sugata, A. et al. (2002). High-throughput screening for GJB2 mutations its clinical application to genetic testing in prelingual deafness screening for GJB2 mutations. Auris, Nasus, Larynx. 29: 231-239.

3. Elit, L., Jack, E., Kwan, E., Baigal, G., and S. Narod. (2001). A unique BRCA1 mutation identified in Mongolia. International Journal of Gynecological Cancer. 11: 241-243.

4. DNA Genotek. (2004). DNA Yield with Oragene. WP002 Rev 1.5 Sept 04.


back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. DNA Stability with Oragene
2. Oragene and Whole Genome Amplification with GenomiPhi
3. DNA Yield with Oragene
4. Oragene is compatible with TaqMan SNP genotyping
5. Serial Analysis of Gene Expression Using ABI PRISM DNA Sequencers and BigDye Terminators
6. Total RNA Purification from Cultured Cells Using the ABI PRISM 6700 Automated Nucleic Acid Workstation and Total RNA Lysis Reagents
7. Rapid, High Throughput Mutation Discovery Using Fluorescent SSCP on the ABI PRISM 3100 and 3100Avant Genetic Analyzers
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:4/29/2016)... CO (PRWEB) , ... April 29, 2016 , ... ... addressing the necessary fundamentals to transform technology into a viable company, CereScan’s CEO, ... growth. Mr. Kelley, a recognized leader and mentor in the Denver area ...
(Date:4/28/2016)... , April 28, 2016 ... company reports the Company,s CEO  was featured in ... Accelerators Enter When VCs Fear To Tread: ... Leader magazine is an essential business ... everything from emerging biotechs to Big Pharmas. Their ...
(Date:4/27/2016)... ... April 27, 2016 , ... Cambridge Semantics, the leading provider of ... that it has been named to The Silicon Review’s “20 Fastest Growing Big Data ... Cambridge Semantics serves the needs of end users facing some of the most complex ...
(Date:4/27/2016)... SILVER SPRING, Md. and RESEARCH ... -- United Therapeutics Corporation (NASDAQ: UTHR ) announced ... Co-Chief Executive Officer, of United Therapeutics will provide an ... Deutsche Bank 41 st Annual Health Care Conference. ... May 5, 2016, at 10:00 a.m. Eastern Time, and ...
Breaking Biology Technology:
(Date:3/18/2016)... --> --> Competitive Landscape Analysis ... Physical infrastructure and Perimeter Surveillance & Detection Systems ... and the continuing migration crisis in the Middle ... led visiongain to publish this unique report, which is crucial ... & security companies in the border security market and the ...
(Date:3/14/2016)... 2016 NXTD ) ("NXT-ID" or the ... announces the airing of a new series of commercials on ... March 21 st .  The commercials will air on Bloomberg ... on the Street show. --> NXTD ) ("NXT-ID" ... commerce market, announces the airing of a new series of ...
(Date:3/10/2016)... , March 10, 2016 ... new market research report "Identity and Access Management Market ... Audit, Compliance, and Governance), by Organization Size, by Deployment, ... 2020", published by MarketsandMarkets, The market is estimated to ... 12.78 Billion by 2020, at a Compound Annual Growth ...
Breaking Biology News(10 mins):