Method for detecting target nucleic acid sequence using cleaved complementary tag fragment and a composition therefor

Abstract

The present invention relates to a method and a composition for detecting a target nucleic acid sequence using a cleaved complementary tag fragment. Specifically, the present invention relates to a method for linking a complementary tag sequence to a PCR primer so that a tagging can be produced by a restriction enzyme during a PCR reaction, diversifying the complementary tag sequence to be linked to each primer by utilizing factors such as length and nucleic acid combination, etc., and distinguishing the target sequence using the same. According to the present invention, a cleaved complementary tag fragment (CCTF) under stringent conditions is a complementary sequence to any sequence at the 5′ end linked to the primer and cannot be formed unless a PCR reaction and a restriction enzyme reaction occur, and the cleaved single strand is formed only when hybridization to the target sequence occurs and a primer extension product complementary to the target sequence is formed, so as to have a higher degree of accuracy secured by reading the cleaved single strand. In addition, the CCTF can be used to identify a plurality of target nucleic acid sequences by selecting various analytical techniques and analysis equipment according to a user's intention. For example, a result can be confirmed rapidly and accurately in genetic testing, identification of organisms in a sample, diagnosis of microbial or viral infection, etc.

What is claimed is: 
     
         1 . A method for forming and identifying a tag used in classifying and analyzing kinds of the target sequences amplified in the Polymerase Chain Reaction, which comprises:
 a) hybridizing a target sequence with a primer comprising a template of a tag for generating the tag, which is a cleaved complementary tag fragment;   b) generating the complementary tag fragment cleaved from the primer by an activity of a restriction enzyme when the amplification procedure is proceeded by the hybridization of a) step and releasing and introducing it into a reaction solution; and   c) identifying the generated cleaved complementary tag fragment through an analyzer to confirm the presence of the target nucleic acid sequence   wherein said primer of a) step comprises a random nucleic acid sequence noncomplementary to a target sequence and has a structure sequentially comprising a restriction enzyme recognition sequence and a nucleic acid sequence complementary to the target sequence.   
     
     
         2 . The method according to  claim 1 , characterized in that the restriction enzyme recognition sequence is the recognition sequence for the restriction enzyme selected from the group consisting of Pho I, PspGI, BstNI, TfiI, ApeKI, TspMI, BstBI, BstEII, BstNI, BstUI, BssKI, BstYI, TaqI, MwoI, TseI, Tsp45I, Tsp509I, TspRI, Tth111I, Nb.BsmI, Nb.BsrDI, NLBspQI, Nt.BstNBI restriction enzymes and Nick restriction enzyme. 
     
     
         3 . The method according to  claim 1 , characterized in that the modified dNTP is inserted in a region of the primer cleaved by a restriction enzyme in order to prevent cleaved by-products other than the cleaved complementary tag fragment from participating in the reaction. 
     
     
         4 . The method according to  claim 3 , characterized in that the modified dNTP inserted in the cleaved region comprises Phosphorothioated dNTP, dNTP comprising 7-Deazapurine, or 2′-O-methyl nucleotide(2′-OMeN) in DNA template. 
     
     
         5 . The method according to  claim 1 , characterized in that the said method analyzes the mass of the cleaved complementary tag fragment to identify the cleaved complementary tag fragment. 
     
     
         6 . The method according to  claim 5 , characterized in that the instrument used for the mass spectrometry is a matrix-assisted laser desorption-ionization-time-of-flight mass spectrometer (MALDI-TOF MS), a Liquid Chromatography Mass Spectrometer, or a High Performance Liquid Chromatography Mass Spectrometer. 
     
     
         7 . The method according to  claim 6 , characterized in that the mass per unit electric charge (m/z) of the cleaved tag fragment used for mass spectrometry is present in the range of from greater than 0 to 10000 Da or less. 
     
     
         8 . The method according to  claim 7 , characterized in that DNA polymerase that the function of adenine addition elongation effect (A tailing) at the 3′ end, being an inherent property of the polymerase is inhibited, is used in order to preserve the mass of a cleaved complementary tag fragment used in mass analysis during the amplification process. 
     
     
         9 . The method according to  claim 1 , characterized in that the fluorescence signal is analyzed by using the oligonucleotide that is tagged by fluorescence and Quencher and has the complementary sequence of the cleaved complementary tag fragment, as the identification method of the cleaved complementary tag fragment. 
     
     
         10 . The method according to  claim 9 , characterized in that the said method analyzes the dissociation temperature and melting peak by varying the inherent dissociation temperature at which the double strand of the oligonucleotide and the cleaved complementary tag fragment are dissociated into a single strand, and identifies the cleaved complementary tag fragment to confirm the presence of the target sequence. 
     
     
         11 . The method according to  claim 9 , characterized in that the said method is made to have different dissociation temperatures to simultaneously analyze two or more kinds of targets through a melting peak analysis in the case of that two or more targets are detected. 
     
     
         12 . The method according to  claim 9 , characterized in that the oligonucleotide is from 5 or more to 50 or less in length. 
     
     
         13 . The method according to  claim 9 , characterized in that the nucleotide at the 3′end of the oligonucleotide is blocked in order to prevent elongation of the base sequence from the oligonucleotide. 
     
     
         14 . The method according to  claim 13 , characterized in that the said method attaches Spacer C3, Phosphat, ddC, or Inverted END to the nucleotide at the 3′end of the oligonucleotide in order to prevent elongation of the base sequence from the oligonucleotide. 
     
     
         15 . The method according to  claim 13 , characterized in that the said method attaches the quencher to the nucleotide at the 3′end of the oligonucleotide is blocked in order to prevent elongation of the base sequence from the oligonucleotide. 
     
     
         16 . The method according  claim 1 , characterized in that the method identifies a causative organism of the sexually transmitted disease, the causative organism of gastrointestinal tract disease, a Human Papilloma virus, a causative organism of the respiratory disease, or a gene type of a single nucleotide polymorphism (SNP). 
     
     
         17 . The method according to  claim 9 , characterized in that the method identifies the complementary tag fragment cleaved by analyzing the cycle threshold (Ct) value of the fluorescence signal of the oligonucleotide.