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:10/11/2017)... ... October 11, 2017 , ... Disappearing forests and increased emissions are the main ... people each year. Especially those living in larger cities are affected by air pollution ... of the most pollution-affected countries globally - decided to take action. , “I knew ...
(Date:10/11/2017)... Florida (PRWEB) , ... October 11, 2017 , ... ... Drug Administration (FDA) has granted orphan drug designation to SBT-100, its novel anti-STAT3 ... for the treatment of osteosarcoma. SBT-100 is able to cross the cell membrane ...
(Date:10/10/2017)... ... October 10, 2017 , ... ... antibody-drug conjugate (ADC) therapeutics, today confirmed licensing rights that give it exclusive ... a technology developed in collaboration with Children’s Hospital Los Angeles (CHLA). ...
(Date:10/10/2017)... ... ... Dr. Bob Harman, founder and CEO of VetStem Biopharma, Inc. ... event entitled “Stem Cells and Their Regenerative Powers,” was held on August 31st, ... was joined by two human doctors: Peter B. Hanson, M.D., Chief of Orthopedic Surgery, ...
Breaking Biology Technology:
(Date:6/14/2017)... 15, 2017  IBM (NYSE: IBM ) is introducing several ... dedicated to developing collaboration between startups and global businesses, taking ... During the event, nine startups will showcase the solutions they ... industries. France is ... with a 30 percent increase in the number of startups ...
(Date:5/6/2017)... RAM Group , Singaporean based ... in biometric authentication based on a novel  ... to perform biometric authentication. These new sensors are based on ... Ram Group and its partners. This sensor will have ... and security. Ram Group is a next generation ...
(Date:4/17/2017)... NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" or the ... Annual Report on Form 10-K on Thursday April 13, 2017 with ... ... Relations section of the Company,s website at http://www.nxt-id.com  under "SEC ... . 2016 Year Highlights: Acquisition ...
Breaking Biology News(10 mins):