title: Isolation of single-stranded DNA from loop-mediated isothermal amplification products.
abstract: Loop-mediated isothermal amplification (LAMP), in which a specific DNA sequence can be directly amplified under isothermal conditions, yields DNA in large quantities of more than 500 microg/ml. We have developed a method to isolate single-stranded DNA fragments from LAMP products that are stem-loop DNAs with several inverted repeats of the target DNA. This method requires the TspRI restriction enzyme, a primer hybridized to the 3' overhanging sequence at its cleavage site, and a DNA polymerase with strand displacement activity. The LAMP products are digested with TspRI and are then extended using the primer, producing the strand-specific DNA fragments. All processes, from LAMP reaction to primer extension, can be carried out at the same temperature. The use of strand-specific DNA would be conducive for detection by hybridization technique such as DNA microarrays.
authors.affiliation: Eiken Chemical Company Ltd., 1381-3, Shimoishigami, Ohtawara, 324-0036, Japan. kentaro_nagamine@eiken.co.jp
authors.forename: Kentaro
authors.initials: K
authors.lastname: Nagamine
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kuzuhara
authors.forename: Tsugunori
authors.initials: T
authors.lastname: Notomi
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D005784:Gene Amplification; D005822:Genetic Vectors; D021141:Nucleic Acid Amplification Techniques; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D010957:Plasmids / Q000235:genetics
chemical_list: D017931:DNA Primers; D004277:DNA, Single-Stranded; D004262:DNA Restriction Enzymes