BRING SANGER SEQUENCING TO THE COMMUNITY CLINICAL LABORATORIES
WORLDWIDE
Sin Hang Lee and Guofan Hong
Milford Hospital and Milford Molecular Diagnostics Laboratory, 2044 Bridgeport Avenue, Milford,
CT 06460, USA
Abstract
One obstacle to implement automated Sanger sequencing as routine tests in developed countries and
as needed accurate diagnostic tools for emerging infectious diseases, such as Ebola, is the labile PCR
reagents, namely the DNA polymerases, the dNTPs and the reverse transcriptases, which must be
kept at -20°C between uses. We have optimized a PCR chemical mix in which these labile
components are stabilized at room temperature for several weeks to 10 months. It depends on a highly processive,
moderately heat-resistant DNA polymerase with a PCR thermocycling not to exceed 85°C [1], and has been successfully
used for preparing DNA sequencing templates in the routine detection and genotyping of human papillomaviruses [2], in
the diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis [3] and in the diagnosis of Lyme borrelioses [4-6].
By adding a reverse transcriptase in this low temperature PCR mix, the newly created RT-PCR mix may be adapted for
screening Ebola patient samples at the sites of outbreak in far-flung places and the PCR amplicons can be transported to
a regional laboratory for DNA sequencing validation.
REFERENCES
[1] Hong G et al. Inter J Mol Sci. 2013; 14:12853-12862.
[2] Lee SH. Methods Mol Biol 2012; 903:65-101.
[3] Lee SH et al. Am J Clin Pathol. 2008; 129:852-859.
[4] Lee SH et al. Am J Clin Path. 2010; 133:569-576.
[5] Lee SH et al. Inter J Mol Sci. 2014; 15:4284-4298.
[6] Lee SH et al. Inter J Mol Sci. 2014; 15:11364-11386.