Journal: Yonago Acta Medica
Article Title: Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Simple and Rapid Detection of Promyelocytic Leukemia–Retinoic Acid Receptor α mRNA
Figure Lengend Snippet: Dde I restriction enzyme digestion of RT-LAMP products. Products of bcr1, bcr2, and bcr3 plasmid DNA amplification were incubated with (+) or without (−) Dde I at 37 °C for 120 min, followed by deactivation at 70 °C for 15 min and separation by 1.5% agarose gel electrophoresis (100 V, 35 min). The amounts of bcr1, bcr2 and bcr3 plasmid DNAs were 8.8, 7.8, and 8.0 × 10 6 copies/tube, respectively. The expected sizes of digested RT-LAMP products are the followings: 468 bp, about 234 bp (stem and loop structure) and 83 bp for bcr1; 232 bp, about 116 bp (stem and loop structure) and 83 bp for bcr2; and 139 bp and 121 bp (merged in Figure) for bcr3. DW, distilled water (negative control); M, 100-bp ladder size marker; neg RNA, IVS-0035 RNA (negative control); RT-LAMP, reverse transcription loop-mediated isothermal amplification.
Article Snippet: The specificity of LAMP was confirmed by restriction enzyme digestion using Dde I (Takara Bio, Otsu, Japan) under the following conditions: 5 μL LAMP product was mixed with 2 μL of 10 × K buffer (Takara Bio), 1 μL Dde I, with distilled water added to obtain a final volume of 20 μL, followed by incubation at 37 °C for 120 min. Dde I was inactivated at 70 °C for 15 min and the digestion products (10-μL aliquots) were mixed with loading buffer (Nippon Gene, Toyama, Japan) and separated by 1.5% agarose gel electrophoresis (100 V, 35 min) with 0.005% ethidium bromide using a Mupid mini gel electrophoresis system (Advance, Tokyo, Japan), with 5 μL Gene Ladder 100 (Nippon Gene) used as a molecular size marker.
Techniques: Plasmid Preparation, Amplification, Incubation, Agarose Gel Electrophoresis, Negative Control, Marker