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:5/25/2016)... , ... May 25, 2016 , ... Scientists at the ... options being tried for mesothelioma may be hampering the research that could lead to ... Click here to read it now. , The team evaluated 98 ...
(Date:5/25/2016)... ... May 25, 2016 , ... ... and Drug Administration (FDA) has granted the company’s orphan drug designation request covering ... orphan drug designation granted by the FDA. , Spinocerebellar ataxia is a ...
(Date:5/24/2016)... , ... May 24, 2016 , ... Last week, Callan ... corporate executives and entrepreneurs, held The Future of San Diego Life Science event at ... Diego life science community attended the event with speakers Dr. Rich Heyman, former CEO ...
(Date:5/23/2016)... ... May 23, 2016 , ... PrecisionAg® Media has released its ... Beyond. The paper outlines the key trends that are creating both opportunities and ... a lot of highs and lows as the precision agriculture market has grown ...
Breaking Biology Technology:
(Date:3/29/2016)... BOCA RATON, Florida , March 29, 2016 /PRNewswire/ ... ("LegacyXChange" or the "Company") LegacyXChange "LEGX" and SelectaDNA/CSI Protect ... Synthetic DNA in ink used in a variety of ... preventing theft. Buyers of originally created collectibles from athletes ... authenticity through forensic analysis of the DNA. ...
(Date:3/21/2016)... , March 22, 2016 ... recognition with passcodes for superior security   ... a leading provider of secure digital communications services, today ... biometric technology and offer enterprise customers, particularly those in ... facial recognition and voice authentication within a mobile app, ...
(Date:3/15/2016)... JERUSALEM , March 15, 2016 ... Jerusalem , the technology-transfer company of the Hebrew University, ... developer of remote sensing technology of various human biological ... funding, raising $2.0 million from private investors. ... technology, based on the detection of electromagnetic emissions from ...
Breaking Biology News(10 mins):