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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
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