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A modification of
conventional sequencing technology that simultaneously determines
DNA sequences from multiple genomic regions can detect sexually
transmitted pathogens.
Nucleotide sequence-based multitarget
identification.
Vinayagamoorthy T, Mulatz K, Hodkinson R.
Journal of Clinical
Microbiology 2003;41:3284-3292.
Summary:
Question
What is the basic concept of MULTIGEN, a modification of conventional
sequencing technology that generates sequences from multiple DNA targets,
and what are the applications for diagnostic testing?
Design
The basic concept, features, and protocols of MULTIGEN technology were
described and applied to the detection of, among other targets, the STD
pathogens N. gonorrhoeae, C. trachomatis, and U.
urealyticum to show the feasibility of MULTIGEN technology for routine
diagnostic testing.
Description of Tests and Diagnostic
Standard
DNA from N. gonorrhoeae, C. trachomatis, and U. urealyticum
was amplified by multiplex PCR using primers and templates from PCR-based
detection kits (Maxim Biotech) to generate amplicons of 298 bp from the
CppB gene, 364 bp from the cryptic plasmid, and 219 bp from the ureB gene,
respectively. The amplified multiple targets were purified and sequenced
by terminator cycle sequencing (ABI Prism BigDye, Applied Biosystems)
using corresponding sequencing primers, and analyzed by capillary
electrophoresis (ABI Prism 310). The sequencing primers were designed to
restrict the length of the sequencing segments to a short stretch (less
than 100 nucleotides) at the 3' end of the amplicon sequenced. Each of the
three sequencing primers, "a, b, and c", corresponding to each
of the three DNA targets (N. gonorrhoeae, C. trachomatis, and U.
urealyticum) in the reaction was modified at the 5' end so that the
molecular weight of primer b was greater than the molecular weights of all
sequences synthesized by primer a, and the molecular weight of primer c
was greater than the molecular weights of all sequences synthesized by
primer b. By using a mixture of sequencing primers of progressively
increasing molecular weights, different sets of truncated DNA molecules
specific to each DNA target were generated and detected simultaneously in
a single lane using capillary electrophoresis.
Main Outcome Measures
Electropherograms showing electrophoretic separation and sequences from
multiple targets were presented.
Main Results
An electropherogram of analyzed data showed 28 nucleotides of U.
urealyticum sequence, 25 nucleotides of N. gonorrhoeae
sequence, and 23 nucleotides of C. trachomatis sequence obtained
from one PCR and one sequencing reaction and migrating in distinct regions
of the sequencing gel.
Authors' Conclusions
MULTIGEN technology can simultaneously
determine, in the same clinical specimen, multiple short DNA sequences
from a variety of targets including N. gonorrhoeae, C. trachomatis
and U. urealyticum.
Source of funding:
Bio-ID Diagnostic, Inc., Saskatoon, Saskatchewan, Canada
For correspondence:
T. Vinayagamoorthy, Bio-ID Diagnostics, Inc., 7, LFK Biotechnology
Complex, 410 Downey Rd., Saskatoon, S7N 4N1 Saskatchewan, Canada. E-mail
address: moorthy@innovationplace.com.
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