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:6/23/2016)... 23, 2016 Houston Methodist Willowbrook Hospital ... Sports Association to serve as their official health ... Methodist Willowbrook will provide sponsorship support, athletic training ... association coaches, volunteers, athletes and families. ... Sports Association and to bring Houston Methodist quality ...
(Date:6/23/2016)... Prostate Cancer Foundation (PCF) is pleased to announce 24 new Young Investigator ... Members of the Class of 2016 were selected from a pool of 128 ... About the Class of 2016 PCF Young Investigators ... ... ...
(Date:6/23/2016)... , June 23, 2016   EpiBiome , a ... $1 million in debt financing from Silicon Valley Bank ... automation and to advance its drug development efforts, as ... facility. "SVB has been an incredible strategic ... services a traditional bank would provide," said Dr. ...
(Date:6/23/2016)... ... 23, 2016 , ... STACS DNA Inc., the sample tracking software company, today ... Laboratory, has joined STACS DNA as a Field Application Specialist. , “I am ... and COO of STACS DNA. “In further expanding our capacity as a scientific integrator, ...
Breaking Biology Technology:
(Date:6/21/2016)... , June 21, 2016 NuData ... the new role of principal product architect and ... the director of customer development. Both will report ... technical officer. The moves reflect NuData,s strategic growth ... response to high customer demand and customer focus ...
(Date:6/9/2016)... 2016 Paris Police Prefecture ... security solution to ensure the safety of people and operations ... the major tournament Teleste, an international technology group ... announced today that its video security solution will be utilised ... up public safety across the country. The system roll-out is ...
(Date:6/2/2016)... 2016 Perimeter Surveillance & Detection ... Physical Infrastructure, Support & Other Service  The ... offers comprehensive analysis of the global Border Security ... revenues of $17.98 billion in 2016. Now: ... leader in software and hardware technologies for advanced video ...
Breaking Biology News(10 mins):