Method for producing RNA-containing probe for detecting a target nucleotide

• US 9,336,349 B2
• Filed: 07/28/2011
• Issued: 05/10/2016
• Est. Priority Date: 07/29/2010
• Status: Active Grant

• Current Assignee: Takara Bio Incorporated (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Bio Incorporated (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Yamamoto, Junko, Inden, Tomoo, Ohba, Toshiharu, Suzuki, Tooru, Mukai, Hiroyuki, Asada, Kiyozo
• Primary Examiner(s): Martinell, James
• Application Number:
  US13/812,242
• Publication Number:
  US 20130122503A1
• Time in Patent Office: 1,748 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 5/00 ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks

Abstract

An object of the present invention is to provide a simple and useful method for producing an RNA-containing probe for detecting a target nucleotide, a simple and useful method, device, and system for processing nucleotide sequence information, and a simple and useful method for detecting a target nucleotide. The present invention provides a method for processing nucleotide sequence information, the method comprising the step of generating partial nucleotide sequences which has 7 to 14 nucleotides and a Tm value of 25 to 40° C. and in which a target nucleotide or a nucleotide adjacent to the target nucleotide is located at a position between 3 and 5 nucleotides from the 3′ or 5′ end. The method according to the present invention is useful for simply and efficiently producing an RNA-containing probe for detecting a target nucleic acid, without the basis of researchers'"'"' experiences or guess, and are extremely useful not only in the field of genetic engineering, but also in the field of medical research.

Claims

1. A method for producing an RNA and DNA-containing probe for use in a method of detecting a target nucleic acid based on whether or not the probe is cleaved by ribonuclease H after the probe is hybridized to the target nucleic acid which comprises the step of preparing a nucleic acid fragment containing RNA and DNA having the same sequence as a partial nucleotide sequence obtained by the RNA nucleotide conversion which is generated by a the method for processing nucleotide sequence information, wherein the method for processing nucleotide sequence information is carried out by a device for processing the nucleotide sequence information and comprises the steps of:

(1) by the device, outputting inputted data of a nucleotide sequence comprising position information of a target nucleotide wherein the inputted data of the nucleotide sequence comprise the target nucleotide, the nucleotide adjacent to the 5′

side of the target nucleotide, and the nucleotide adjacent to the 3′

side of the target nucleotide;

(2) by the device, generating data of a nucleotide sequence comprising the position information of the target nucleotide which is complementary to the outputted data of the nucleotide sequence comprising the position information of the target nucleotide;

(3) by the device, generating data of a nucleotide sequence which is obtained by converting the target nucleotide or the nucleotide adjacent to the target nucleotide in the nucleotide sequence into an RNA, in the data of the nucleotide sequence comprising the position information of the target nucleotide or the nucleotides each adjacent to the target nucleotide and the data of the complementary nucleotide sequence thereof; and

(4) by the device, generating data of a partial nucleotide sequence comprising the position information of the target nucleotide or the nucleotides each adjacent to the target nucleotide, from the data of the nucleotide sequence and the data of the complementary nucleotide sequence thereof obtained by the RNA nucleotide conversion, wherein

(a) the number of nucleotides in the partial nucleotide sequence is 7 to 14,

(b) the Tm value of the partial nucleotide sequence is 25 to 40°

C., wherein the Tm value is calculated under conditions having a DNA concentration of 500nM and a salt concentration of 50 mM, and

(c) the target nucleotide or the nucleotide adjacent to the target nucleotide in the nucleotide sequence is located at a position between 3 and 5 nucleotides from the 3′

end of the nucleotide sequence or between 3 and 5 nucleotides from the 5′

end of the nucleotide sequence,

(5) by the device, using the data of the partial nucleotide sequence obtained by the RNA nucleotide conversion to generate data of a nucleotide sequence of an RNA and DNA-containing probe used in a method of detecting a target nucleic acid based on whether or not the probe is cleaved by ribonuclease H after the probe is hybridized to target nucleic acid, and

wherein the method more efficiently generates the data of the nucleotide sequence of an RNA and DNA-containing probe wherein the probe more efficiently detects the target nucleic acid and detects the target nucleic acid with a high level of specificity.

2. A method for detecting a target nucleotide, which comprises the steps of:

(1) producing a RNA and DNA-containing probe for detecting a target nucleotide by the method according to claim 1,

(2) hybridizing to nucleic acids in a sample the RNA and DNA-containing probe

(3) applying ribonuclease H treatment to the RNA and DNA-containing probe for detecting the target nucleotide, after the hybridization step; and

(4) detecting whether or not the RNA and DNA-containing probe for detecting the target nucleotide has been cleaved by the ribonuclease H.

4. A method for detecting a target nucleotide, which comprises the steps of:

(1) producing a RNA and DNA-containing probe for detecting a target nucleotide by the method according to claim 3,

(2) hybridizing to nucleic acids in a sample the RNA and DNA-containing probe;

(3) applying ribonuclease H treatment to the RNA and DNA-containing probe for detecting the target nucleotide, after the hybridization step; and

(4) detecting whether or not the RNA and DNA-containing probe for detecting the target nucleotide has been cleaved by the ribonuclease H.

6. A method for detecting a target nucleotide, which comprises the steps of:

(1) producing a RNA and DNA-containing probe for detecting a target nucleotide by the method according to claim 5,

(2) hybridizing to nucleic acids in a sample the RNA and DNA-containing probe;

(3) applying ribonuclease H treatment to the RNA and DNA-containing probe for detecting the target nucleotide, after the hybridization step; and

(4) detecting whether or not the RNA and DNA-containing probe for detecting the target nucleotide has been cleaved by the ribonuclease H.

3. A method for producing an RNA and DNA-containing probe for use in a method of detecting a target nucleic acid based on whether or not the probe is cleaved by ribonuclease H after the probe is hybridized to the target nucleic acid, which comprises the step of preparing a nucleic acid fragment containing RNA and DNA having the same sequence as a partial nucleotide sequence obtained by the RNA nucleotide conversion which is generated by a the method for processing nucleotide sequence information, wherein the method for processing nucleotide sequence information is carried out by a device for processing the nucleotide sequence information and comprises the steps of:

(1) by the device, outputting inputted data of a nucleotide sequence comprising position information of a target nucleotide wherein the inputted data of the nucleotide sequence comprise the target nucleotide, the nucleotide adjacent to the 5′

side of the target nucleotide, and the nucleotide adjacent to the 3′

side of the target nucleotide;

(2) by the device, generating data of a nucleotide sequence comprising the position information of the target nucleotide which is complementary to the outputted data of the nucleotide sequence comprising the position information of the target nucleotide;

(3) by the device, generating data of a partial nucleotide sequence comprising the position information of the target nucleotide or the nucleotides each adjacent to the target nucleotide, from the outputted data of the nucleotide sequence and the generated data of the complementary nucleotide sequence, wherein

(a) the number of nucleotides in the partial nucleotide sequence is 7 to 14,

(b) the Tm value of the partial nucleotide sequence is 25 to 40°

C., wherein the Tm value is calculated under conditions having a DNA concentration of 500 nM and a salt concentration of 50 mM, and

(c) the target nucleotide or the nucleotide adjacent to the target nucleotide in the nucleotide sequence is located at a position between 3 and 5 nucleotides from the 3′

end of the nucleotide sequence or between 3 and 5 nucleotides from the 5′

end of the nucleotide sequence; and

(4) by the device, generating data of a partial nucleotide sequence which is obtained by converting the target nucleotide or the nucleotide adjacent to the target nucleotide in the nucleotide sequence into an RNA, in the data of the partial nucleotide sequence comprising the position information of the target nucleotide or the nucleotides each adjacent to the target nucleotide,

(5) by the device, using the data of the partial nucleotide sequence obtained by the RNA nucleotide conversion to generate data of a nucleotide sequence of an RNA and DNA-containing probe used in a method of detecting a target nucleic acid based on whether or not the probe is cleaved by ribonuclease H after the probe is hybridized to target nucleic acid, and

wherein the method more efficiently generates the data of the nucleotide sequence of an RNA and DNA-containing probe wherein the probe more efficiently detects the target nucleic acid and detects the target nucleic acid with a high level of specificity.

5. A method for producing an RNA and DNA-containing probe for use in a method of detecting a target nucleic acid based on whether or not the probe is cleaved by ribonuclease H after the probe is hybridized to the target nucleic acid, which comprises the step of preparing a nucleic acid fragment containing RNA and DNA having the same sequence as a partial nucleotide sequence obtained by the RNA nucleotide conversion which is generated by a the method for processing nucleotide sequence information wherein the method for processing nucleotide sequence information is carried out by a device for processing the nucleotide sequence information and comprises the steps of:

(1) by the device, outputting inputted data of a nucleotide sequence comprising position information of a target nucleotide wherein the inputted data of the nucleotide sequence comprise the target nucleotide, the nucleotide adjacent to the 5′

side of the target nucleotide, and the nucleotide adjacent to the 3′

side of the target nucleotide;

(2) by the device, generating data of a nucleotide sequence comprising the position information of the target nucleotide which is complementary to the outputted data of the nucleotide sequence comprising the position information of the target nucleotide;

(3) by the device, making at least one determination of the following determinations;

(a) a determination as to whether or not the target nucleotide is a purine nucleotide,

(b) a determination as to whether or not the nucleotide adjacent to the 3′

side of the target nucleotide is a purine nucleotide, and

(c) a determination as to whether or not the nucleotide adjacent to the 5′

side of the target nucleotide is a purine nucleotide, for the outputted data of the nucleotide sequence and the generated data of the complementary nucleotide sequence, whereby one nucleotide that is a purine nucleotide is determined as an RNA position from the outputted data of the nucleotide sequence and the generated data of the complementary nucleotide sequence, an outputting data of a nucleotide sequence comprising the RNA position information;

(4) by the device, generating data of a partial nucleotide sequence comprising the RNA position information from the outputted data of the nucleotide sequence comprising the RNA position information, wherein

(d) the number of nucleotides in the partial nucleotide sequence is 7 to 14,

(e) the Tm value of the partial nucleotide sequence is 25 to 40°

C., wherein the Tm value is calculated under conditions having a DNA concentration of 500 nM and a salt concentration of 50 mM, and

(f) the one nucleotide indicated at the RNA position is located at a position between 3 and 5 nucleotides from the 3′

end of the nucleotide sequence or between 3 and 5 nucleotides from the 5′

end of the nucleotide sequence; and

(5) by the device, generating data of a partial nucleotide sequence which is obtained by converting the nucleotide determined as the RNA position into an RNA, in the data of the nucleotide sequence comprising the RNA position information or the partial nucleotide sequence comprising the RNA position information,

(6) by the device, using the data of the partial nucleotide sequence obtained by the RNA nucleotide conversion to generate data of a nucleotide sequence of an RNA and DNA-containing probe used in a method of detecting a target nucleic acid based on whether or not the probe is cleaved by ribonuclease H after the probe is hybridized to target nucleic acid, and

wherein the method more efficiently generates the data of the nucleotide sequence of an RNA and DNA-containing probe wherein the probe more efficiently detects the target nucleic acid and detects the target nucleic acid with a high level of specificity.