Loop-mediated isothermal amplification of a single DNA molecule in polyacrylamide gel-based microchamber.
Loop-mediated isothermal amplification (LAMP) is an original nucleic acid amplification method established by Notomi et al. LAMP is performed under isothermal condition, employing only a basic reaction protocol and minimal supporting electronics. These requirements prove to be viable for exploring the avenues to down-scale this biological reaction for Lab-on-a-chip application. Hence here, we developed a novel technique for fluorescent imaging of LAMP at a single molecule level. The experiment was conducted in a polyacrylamide (PAA) gel-based microchamber where a single DNA template, freely suspended in a solution containing primers and polymerase was initially encapsulated. In order to activate the amplification reaction, a microheater regulated by an automatic computerized feedback system was used for localized heating. This microchamber-based approach for LAMP demonstrated the effective exploitation of minute amount of templates and primers, and the overall reduction in LAMP detection time. An average efficiency of 80% was evaluated for conducting DNA amplification after 50 min of incubation at 65 degrees C. As the total time for reaction including detection can be completed in less than 1 h, this one-step, direct observation method displays the potential as a simple alternative to conventional techniques for genetic analysis and diagnosis in the clinical laboratory.
Lam
Liza
L
The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan. lizalam@sanken.osaka-u.ac.jp
Sakakihara
Shouichi
S
Ishizuka
Koji
K
Takeuchi
Shoji
S
Arata
Hideyuki F
HF
Fujita
Hiroyuki
H
Noji
Hiroyuki
H
D000180:Acrylic Resins / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D006358:Hot Temperature; D008832:Microchemistry / Q000295:instrumentation*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D009930:Organic Chemicals / Q000378:metabolism; D013698:Templates, Genetic; D013997:Time Factors
D000180:Acrylic Resins; D017931:DNA Primers; D004279:DNA, Viral; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C098798:SYBR Green II; C016680:polyacrylamide gels; D004247:DNA