Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o
Reexamination Certificate
1999-04-15
2001-07-17
Whisenant, Ethan (Department: 1655)
Chemistry: analytical and immunological testing
Heterocyclic carbon compound
Hetero-o
C536S023100, C536S024300, C536S024330, C435S006120, C435S091100
Reexamination Certificate
active
06261846
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to special and novel probes, a gene amplifying method using cross (alternate) binding of the special probes, and a method of directly detecting a target gene and a method of detecting antigen/antibody using the gene amplifying method.
2. Description of the Related Art
In recent years, a method of amplifying a gene using a DNA polymerase, represented by a polymerase chain reaction method (hereinafter called the “PCR method”), a method of amplifying a gene by hybridizing polymer DNA having previously branched single-stranded DNA (hereinafter called the “branched DNA probe method”), and so on have been developed for detecting a very small amount of target gene.
The amplification of gene by the PCR method involves annealing hybridize) a primer complementary to a target gene (or a primer), and amplifying the gene through rise and fall of temperature using a thermostable DNA polymerase. Disadvantageously, this method takes a long time from amplification to detection of a gene, and is expensive. In addition, the amplification efficiency and specificity may vary depending on the design of a primer complementary to the target gene.
The amplification of gene by the branched DNA probe method, on the other hand, involves previously synthesizing a branched polymer single-stranded DNA probe, and hybridizing the branched polymer single-stranded DNA probe to a target gene to detect the target gene. However, the hybridization of the branched polymer single-stranded DNA probe to the target gene takes a long time because the branched DNA probe is a polymer. In addition, the branched polymer single-stranded DNA is limited in size, so that the detection of the target gene is also limited.
OBJECT AND SUMMARY OF THE INVENTION
The present invention has been made in view of the problems of the prior art mentioned above, and its object is to provide a method of efficiently amplify a gene without using enzyme or branched DNA.
To solve the problem mentioned above, a gene amplifying method according to the present invention comprises the steps of providing a plurality of pairs of probes each composed of three or more portions complementary to such portions of the other probe; and hybridizing said pairs of probes such that they cross in alternation to form a double-stranded polymer.
The pair of probes for use in the gene amplifying method, may be two DNA probes, a DNA probe and an RNA probe, two RNA probes, two PNA (Peptide Nucleic Acid or Polyamide Nucleic Acid) probes, a PNA probe and a DNA probe, or a PNA probe and an RNA probe.
The pair of probes are structured such that complementary portions at three or more sites are each hybridized in a specific manner without fail during one-to-one hybridization.
A target gene detecting method according to the present invention, in a first aspect, comprises the steps of providing a pair of probes, one of which has a gene in one portion thereof complementary to a target gene, hybridizing a plurality of said pairs of probes with the target gene by the gene amplifying method described above to form a double-stranded polymer, and amplifying the probes to detect the target gene.
An antigen/antibody detecting method according to the present invention, in a first aspect, comprises the steps of providing a pair of probes, one of which has a gene in one portion thereof complementary to a target gene bound to antigen/antibody, hybridizing a plurality of said pairs of probes with the target gene by the gene amplifying method described above to form a double-stranded polymer, and detecting antigen/antibody.
The target gene detecting method according to the present invention, in a second aspect, comprises the steps of providing a pair of probes and another probe complementary to a gene in a portion of one of said pair of probes and to a target gene, previously hybridizing said other probe to the target gene, hybridizing said pairs of probes with the target gene by the gene amplifying method described above to form a double-stranded polymer, and amplifying the probes to detect the target gene.
The antigen/antibody detecting method according to the present invention, in a second aspect, comprises the steps of providing a pair of probes and another probe complementary to a gene in a portion of one of said pair of probes and to a target gene bound to antigen/antibody, previously hybridizing said other probe to the target gene, and hybridizing said pairs of probes with the target gene by the gene amplifying method described above to form a double-stranded polymer, and detecting the antigen/antibody.
In the target gene detecting method, an intercalating dye such as ethidium bromide, Oligreen, SYBR Green or the like may be bound to the probes amplified by the gene amplifying method to detect an amplified polymer through fluorescence.
In the antigen/antibody detecting method according to the present invention, an intercalating dye such as ethidium bromide, Oligreen, SYBR Green or the like may be bound to the probes amplified by the gene amplifying method to detect the target gene bound to antigen/antibody or the like through fluorescence.
In the target gene detecting method according to the present invention, as a marker material for detection, a donor fluorescent dye and an acceptor fluorescent dye utilizing fluorescent resonance energy transfer (FRET) such as radioisotope such as
125
I,
32
P or the like, light emitting and coloring materials such as digoxigenin, acridine ester or the like, alkali phosphatase for utilizing a light emitting material such as dioxyethane or the like, and fluorescent materials such as 4-methyl umbelliferil phosphate or the like, and biotin for utilizing fluorescent, light emitting, coloring materials or the like bound to avidin, may be previously added to the probes to be amplified by the gene amplifying method to detect the target gene.
In the antigen/antibody detecting method according to the present invention, as a marker material for detection, a donor fluorescent dye and an acceptor fluorescent dye utilizing fluorescent resonance energy transfer (FRET) such as radioisotope such as
125
I,
32
P or the like, light emitting and coloring materials such as digoxigenin, acridine ester or the like, alkali phosphatase for utilizing a light emitting material such as dioxyethane or the like, and fluorescent materials such as 4-methyl umbelliferil phosphate or the like, and biotin for utilizing fluorescent, light emitting and coloring materials or the like bound to avidin, may be previously added to a pair of probes to be amplified by the gene amplifying method to detect the antigen/antibody.
Each of said probes according to the present invention is composed of three or more portions complementary to such portions of the other probe.
The pair of probes may be two DNA probes, a DNA probe and an RNA probe, two RNA probes, two PNA probes, a PNA probe and a DNA probe, or a PNA probe and an RNA probe.
A double-stranded polymer according to the present invention is formed by using a plurality of the pair of probes, and hybridizing them such that they cross in alternation.
The DNA probe is composed of single-stranded fragments mainly consisting of phosphoric acid, saccharum and bases (adenine, thymine, guanine and cytosine), while the RNA probe is composed of single-stranded fragments mainly consisting of bases which are adenine, uracil, guanine and cytosine. PNA has a structure in which the skeleton of “phosphoric acid and saccharum” in DNA is replaced by an “N-(2-aminoethyl) glycine derivative” and has the same components of bases as DNA and RNA.
The length of the probes for use in the methods mentioned above ranges from 10 bases to 1,000 bases, and most preferably from 10 bases to 100 bases.
The pair of probes are not particularly limited in the type of bases in the mutually complementary portions. In addition, the lengths of the complementary portions existing in one probe may be equal or different.
The number of probes hybridized to form a double-stranded polymer for use in t
Lu Frank
Sanko Junyaku Co. Ltd.
Wenderoth , Lind & Ponack, L.L.P.
Whisenant Ethan
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