METHOD FOR TESTING A MUTANT GENE THROUGH REAL TIME POLYMERASE CHAIN REACTION USING INHIBITION OF 5'-FLAP ENDONUCLEASE ACTIVITY
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Abstract
The present invention relates to a method for detecting single nucleotide polymorphism (SNP) using a feature that the 5′-flap endonuclease (FEN) activity of DNA polymerase is inhibited when a probe complementarily binds to the end of a polymerase chain reaction (PCR) product. More specifically, the present invention relates to a novel method wherein it was verified that, when a probe used for a real-time PCR complementarily binds to the end site of a PCR product, the 5′-FEN activity of thermostable DNA polymerase to the probe is inhibited, and thus when such a feature is used to make a design such that an SNP site to be detected is located at the 5′-end site of the probe, the 5′-flap formation is induced according to the allele, thereby allowing effective SNP detection.
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Citations
32 Claims
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1-17. -17. (canceled)
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18. A method for testing for a mutant gene by real time polymerase chain reaction using a DNA polymerase comprising:
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a) preparing step for template DNA containing at least a mutation region; b) preparing step for forward primer and reverse primer to amplify the mutation region; c) synthetic step for a primer wherein the 5′
-end region of the probe capable of hybridizing to a polymerase chain reaction product can bind to the mutation region of the polymerase chain reaction product, and has a continuous or non-continuous unmatched structure of one or more bases, and the 5′
-end of the probe bound to a polymerase chain reaction product is within 24-38 bases from a 5′
-end of the polymerase chain reaction product;d) amplifying step for the template DNA by adding the primers and the probe, and a DNA polymerase to the template DNA; and e) identifying step for the mutation gene of the template DNA by investigating the PCR amplification plot. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26)
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27. A real time polymerase chain reaction kit for testing for a mutant gene, comprising:
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a template DNA;
a forward primer;
a reverse primer;
a probe; and
a thermostable DNA polymerase,Wherein a 5′
-end of the probe bound to the polymerase chain reaction product has a continuous or non-continuous unmatched structure of one or more bases, and the 5′
-flap endonuclease activity of the thermostable DNA polymerase is inhibited by placing the 5′
-end of the probe bound to the polymerase chain reaction product within 24-38 bases from a 5′
-end of the polymerase chain reaction product. - View Dependent Claims (28, 29, 30, 31, 32)
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Specification