Toyo Boseki Kabushiki Kaisha

Shuji Shibata

Yutaka Takarada

Yoshihisa Kawamura

Hiroaki Inoue

Toshiya Aono

In a method for amplifying the specific nucleic acid sequence, a highly specific amplification which has low possibility of non-specific hybridization can be carried out. Highly stable reagents, the activities of which do not decrease in case of supply and storage, are also provided. Thermostable enzymes are used as RNA dependent DNA polymerase, DNA dependent DNA polymerase, DNA dependent RNA polymerase and ribonuclease H, which are required for the amplification system based on replicated RNA. Especially, it is preferable that a thermostable enzyme derived from,which has RNA dependent DNA polymerase activity, DNA dependent DNA polymerase activity and ribonuclease H activity, and thermostable DNA dependent RNA polymerase are used together. By this method, inactivation of enzymes are prevented by using thermostable enzymes, and the amplification can be carried out without sequential addition of enzymes.

Method for amplifying and detecting of target nucleic acid sequence using thermostable enzyme

In a method for amplifying the specific nucleic acid sequence, a highly specific amplification which has low possibility of non-specific hybridization can be carried out. Highly stable reagents, the activities of which do not decrease in case of supply and storage, are also provided. Thermostable enzymes are used as RNA dependent DNA polymerase, DNA dependent DNA polymerase, DNA dependent RNA polymerase and ribonuclease H, which are required for the amplification system based on replicated RNA. Especially, it is preferable that a thermostable enzyme derived from Thermus thermophilus which has RNA dependent DNA polymerase activity, DNA dependent DNA polymerase activity and ribonuclease H activity, and thermostable DNA dependent RNA polymerase are used together. By this method, inactivation of enzymes are prevented by using thermostable enzymes, and the amplification can be carried out without sequential addition of enzymes.

Method for amplifying and detecting of target nucleic acid sequence

An oligonucleotide is provided, which has at least a base sequence A, which is 10-30 nucleotides in length and homologous to a part of a specific nucleic acid sequence to be amplified, and a base sequence B, which is 10-30 nucleotides in length and complementary to a sequence 3' to the part of the specific nucleic acid sequence to be amplified. Sequences A and B are separated by a spacer region of 0-20 nucleotides. The oligonucleotide is used in an amplification method, wherein the oligonucleotide is mixed with a sample containing a specific nucleic acid sequence to be amplified and allowed to anneal to the specific nucleic acid sequence in the sample. The annealed oligonucleotide acts as a primer in an elongation reaction, whereas any nonannealed oligonucleotide is decomposed, except for at least part of the base sequence A. The elongation product is then denatured to a single strand for use as a template to which the remaining oligonucleotide anneals to form a primer for subsequent elongation. Also provided is a detection method, which allows for detection of a specific nucleic acid sequence in a sample, as well as reagent kits for use in the amplification and detection methods.

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Otsu, Japan

Shuji Shibata