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:10/10/2017)... , Oct. 10, 2017 International research firm Parks ... Strategy, will speak at the TMA 2017 Annual Meeting , October ... trends in the residential home security market and how smart safety and ... Parks ... "The residential ...
(Date:10/10/2017)... ... October 10, 2017 , ... The Pittcon Program ... honoring scientists who have made outstanding contributions to analytical chemistry and ... 2018, the world’s leading conference and exposition for laboratory science, which will be ...
(Date:10/9/2017)... ... , ... At its national board meeting in North Carolina, ARCS® Foundation ... of Physics and Astronomy, has been selected for membership in ARCS Alumni Hall ... 2015 Breakthrough Prize in Fundamental physics for the discovery of the accelerating expansion of ...
(Date:10/7/2017)... Phoenix, Arizona (PRWEB) , ... ... ... than 15 years’ experience providing advanced instruments and applications consulting for microscopy ... the in-house expertise in application consulting, Nanoscience Analytical offers a broad range ...
Breaking Biology Technology:
(Date:8/23/2017)... , Aug. 23, 2017  The general public,s help is being enlisted ... bacteria that live in and on the human body –and are believed ... The Microbiome Immunity ... human microbiome, starting with the gut. The project's goal is to help ... credit: IBM ...
(Date:6/14/2017)... -- IBM (NYSE: IBM ) is introducing several innovative partner ... developing collaboration between startups and global businesses, taking place in ... event, nine startups will showcase the solutions they have built ... France is one of ... 30 percent increase in the number of startups created between ...
(Date:4/24/2017)... , April 24, 2017 Janice ... partner with  Identity Strategy Partners, LLP (IdSP) , ... or without President Trump,s March 6, 2017 ... Entry , refugee vetting can be instilled with greater ... (Right now, all refugee applications are suspended by ...
Breaking Biology News(10 mins):