TH55152A - Methods of nucleic acid synthesis. - Google Patents

Abstract

DC60 (06/01/55) This present invention relates to a novel oligonucleotide and a method for synthesizing nucleic acids using the same as the primer. The oligonucleotide is placed on the 5' side of the primer with a nucleotide sequence almost identical to that of the region synthesized using this primer as the synthesis origin. This present invention realizes the synthesis of nucleic acids based on isothermal reactions with the simple fabrication of various reagents. Furthermore, this present invention provides a highly specific method for nucleic acid synthesis based on this method. This present invention relates to a novel oligonucleotide and a method for synthesizing nucleic acids using the same as the primer. The oligonucleotide is placed on the 5' side of the primer with a nucleotide sequence almost identical to that of the region synthesized using this primer as the synthesis origin. The region synthesized by this primer serves as the synthesis origin. The present invention realizes the synthesis of nucleic acids based on isothermal reactions with the simple fabrication of various reagents. Furthermore, the present invention provides a highly specific method for nucleic acid synthesis based on this method.

Claims (8)

1. A complete set for the synthesis of nucleic acids containing complementary chains linked alternately in a single chain consisting of the following components: i) A first oligonucleotide containing at least two regions, F2 and Flc, where Flc is fused to the 5' side of F2, and where F2 is a region whose nucleotide sequence complements the custom F2c region in a specific nucleotide sequence template nucleic acid, and Flc is a region whose nucleotide sequence is almost identical to the Flc region located at the 5' side of the F2c region in the specific nucleotide sequence template nucleic acid. ii) A second oligonucleotide containing at least two regions, R2 and Rlc, where Rlc is fused to the 5' side of R2, and where R2 is a region whose nucleotide sequence complements the custom R2c region in the complementary chain. Synthesis is performed using the first oligonucleotide as the synthesizing origin, which contains a specific nucleotide sequence, and Rlc is a region with a nucleotide sequence almost identical to the Rlc region located at the 5' side of the R2c region in the associated chain with the specific nucleotide sequence. iii) The first outer primer containing a nucleotide sequence compatible with the F3c region located at the 3' side of the F2c region in the nucleic acid serving as a template. iv) The DNA polymerase that catalyzes the strand substitution type of associated chain synthesis. v) The nucleotide that serves as the substrate for component iv) and vi) The second outer primer containing a nucleotide sequence compatible with the R3c region located at the 3' side of the customized R2c in the associated chain, which is synthesized using the first oligonucleotide as the synthesizing origin. 2. A complete kit as described in item 1, containing a detector for detecting the products of nucleic acid synthesis reactions. 3. The method of synthesizing nucleic acid molecules consists of: a) adding components i) to vi) of the complete set according to point 1 to a single-stranded or double-stranded nucleic acid serving as a template; and b) incubating the mixture of step a) at a temperature at which the nucleotide sequences comprising the first oligonucleotide and the second oligonucleotide can form stable base pairs with their corresponding nucleotide sequences while the enzyme activity can be maintained. 4. The method described in point 3, where the mixture also includes a regulator for the melting temperature. 5. The method described in point 4, where the regulator for the melting temperature is betaine. 6. The method described in point 5, where 0.2 to 3.0 molar betaine is present. 7. Method 3, where the mixture also includes a detector for detecting the products of nucleic acid synthesis, which was prepared using the method in step 3. 8. The method described in point 3, where the template nucleic acid is RNA, and DNA polymerase has reverse transcriptase activity.