Isolation of single-stranded DNA from loop-mediated isothermal amplification products.
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.
Nagamine
Kentaro
K
Eiken Chemical Company Ltd., 1381-3, Shimoishigami, Ohtawara, 324-0036, Japan. kentaro_nagamine@eiken.co.jp
Kuzuhara
Yoko
Y
Notomi
Tsugunori
T
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
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004262:DNA Restriction Enzymes