Real-time turbidimetry of LAMP reaction for quantifying template DNA.
Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the synthesis of large amounts of DNA in a short period of time with high specificity. As the LAMP reaction progresses, the reaction by-product pyrophosphate ions bind to magnesium ions and form a white precipitate of magnesium pyrophosphate. We designed an apparatus capable of measuring the turbidity of multiple samples simultaneously while maintaining constant temperature to conduct real-time measurements of the changes in the turbidity of LAMP reactions. The time (Tt) required for the turbidity of the LAMP reaction solution to exceed a given value was dependent on the quantity of the initial template DNA. That is, a graph with the plot of Tt versus the log of the amount of initial template DNA was linear from 2 x 10(3) copies (0.01 pg/tube) to 2 x 10(9) copies (100 ng/tube) of template DNA. These results indicate that real-time turbidity measurements of the LAMP reaction permit the quantitative analysis of minute amounts of nucleic acids present in a sample, with a high precision over a wide range, using a simple apparatus reported in this study.
Mori
Yasuyoshi
Y
Eiken Chemical Co., Ltd., Biochemical Research Laboratory, 1381-3 Shimoishigami, Ohtawara, Tochigi 324-0036, Japan. yasuyoshi_mori@eiken.co.jp
Kitao
Masataka
M
Tomita
Norihiro
N
Notomi
Tsugunori
T
D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D009391:Nephelometry and Turbidimetry / Q000295:instrumentation / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D013696:Temperature; D013698:Templates, Genetic; D013997:Time Factors
D004247:DNA