Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-04-27
2001-05-01
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06225056
ABSTRACT:
DESCRIPTION
1. Technical Field
This invention relates to solid phases for the detection of target nucleic acids, their preparation methods, and methods of detecting target nucleic acids employing the same.
2. Background Art
With the advent of research developments in recent years, a variety of biological information has come to being derivable from gene sequences. Consequently, detection of the genes (which correspond to the specified polynucleotide sequences of target nucleic acids) has enabled the diagnosis of diseases, susceptibility against drugs, compatibility in organ transplantation and the like in the medical field; it has enabled the detection and identification of diverse pathogens responsible for food poisoning in food science.
In order to detect such specified polynucleotide sequences, hybridization methods are generally performed using probes that comprise sequences complementary to those to be detected. Since the sequences to be detected vary widely in accordance with the purposes of detection, probes having a variety of sequences in accordance with those purposes are employed in the detection. Also, there may be cases where reactions with from tens to a few hundreds of probes have to be examined at one time to confirm the presence of plural sequences in a single sample.
Regarding the simple technique of examining reaction with a large number of probes as described, generally known is a method to use a solid phase such as a filter on which probes have been immobilized.
FIG. 1
schematically illustrates a method where a probe immobilized on the solid phase is used to hybridize to the target nucleic acid and to detect it. This method arranges plural probes on a single solid phase at certain distances; thereby, it allows the simultaneous detection of the reaction with many probes at one time even if a small amount of the sample is used. However, the method employing probes immobilized on the solid phase generally suffers from low efficiencies in reaction with the target nucleic acids because the probes are subjected to restrictions in their movement due to the immobilization, and in addition, it has a difficulty in detection with high sensitivity because background signals arising from the nonspecific adsorption of the sample to the solid phase are generated. Furthermore, mutual binding between nucleic acids themselves by virtue of hybridization takes place even if there are a few mismatches between the sequences and, as a result, the poor ability to recognize sequences is named as a problem common in the hybridization methods.
The oligonucleotide ligation assay is disclosed in Proc. Natl. Acad. Sci. USA (1990) 87, 8923-8927, which is a method to improve the sensitivity and the ability to recognize sequences in the hybridization methods. This method is schematically shown in FIG.
2
. Two probes are reacted with the target nucleic acid: in the two probes a group binding to the solid phase is introduced into one of the two nucleic acids capable of sequentially hybridizing with a target sequence and a group useful for detection is introduced into the other. Then only the correctly hybridized probe is ligated by addition of a ligase. Differing from the methods where probes have been immobilized in advance, these reactions are very efficient since they are conducted in a liquid phase where molecules freely move; further, they enjoy higher sequence recognition than the detection methods solely relying on hybridization, since the ligation reaction by the action of ligase proceeds in such a strict manner that it does not take place if there is disparity in only one base. The probe ligated by the action of ligase can easily be separated by being bound to the solid phase and is detected through the group that has been introduced for the purpose of detection. However, where the sequences to be detected vary diversely, this method compels a technician himself to add different probes depending on the sequences to be detected and, as a result, gives unnecessary tension to the technician, thus likely inducing erroneous additions or the like. Moreover, when plural probes are reacted at the same time, it is difficult to identify as to which sequences correspond to the probes having been detected.
Accordingly, there is a need for the simple method which uses probes arranged on a solid phase in addition to only a small amount of sample and which allows the detection of the reaction of many probes with high sensitivity and great ability of sequence recognition.
DISCLOSURE OF INVENTION
An objective of this invention is to solve the above-mentioned problems and to provide a solid phase for the detection of a target nucleic acid for the purpose of detecting a wide variety of target nucleic acids in a sample simultaneously with more ease, greater rapidity, and high accuracy or for the detection of a large number of base sequence groups contained in a target nucleic acid simultaneously with high accuracy, a preparation method thereof, and a method of detecting a target nucleic acid.
As a result of thorough investigations, the present inventors have found: a solid phase for the detection of a target nucleic acid which enables the detection of a wide variety of target nucleic acids in a sample simultaneously with more ease, greater rapidity, and high sensitivity; a preparation method thereof; and a method of detecting a target nucleic acid using said solid phase.
Specifically, this invention provides a solid phase for the detection of a target nucleic acid comprising a pair of probes, said pair of probes having a base sequence capable of sequentially hybridizing with a specified polynucleotide sequence of said target nucleic acid and being immobilized on said solid phase through a linker portion in such a manner as to occupy a restricted spatial arrangement so that the probes can be ligated by an enzyme when the probes sequentially hybridize with the specified polynucleotide sequence of said target nucleic acid.
Also, the invention provides the solid phase for the detection of a target nucleic acid as described above, characterized being produced by the step wherein the pair of probes immobilized through the linker portion in such a manner as to occupy a restricted spatial arrangement hybridizes with said target nucleic acid to form a complex.
Also, the invention provides the solid phase for the detection of a target nucleic acid as described above, wherein at least one of the pair of probes is further provided with a base sequence capable of hybridizing with a padlock probe.
Further, the invention provides a method of preparing a solid phase for the detection of a target nucleic acid, said method comprising the steps of:
(1) hybridizing a pair of probes with the target nucleic acid to form a complex, said pair of probes having a base sequence capable of sequentially hybridizing with a specified polynucleotide sequence of the target nucleic acid;
(2) immobilizing the pair of probes having formed the complex, on a surface of the solid phase through a linker portion; and
(3) denaturing and removing the target nucleic acid.
Also, the invention provides the method of preparation as described above, wherein the linker portion is formed by a binding reaction of biotin with avidin or streptavidin.
Also, the invention provides the method of preparation as described above, wherein at least one of the pair of probes is further provided with a base sequence capable of hybridizing with a padlock probe.
Further, the invention provides a solid phase for detection, characterized by being produced by the method of preparation as described above.
Still further, the invention provides a method of detecting a target nucleic acid comprising the steps of:
(1) hybridizing a solid phase for the detection of a target nucleic acid with the target nucleic acid to form a complex, said solid phase comprising a pair of probes, said pair of probes having a base sequence capable of sequentially hybridizing with a specified polynucleotide sequence of said target nucleic acid and being immobilized on said solid phase thro
Abe Satoshi
Sato Yoshihiro
Laboratory of Molecular Biophotonics
Leydig , Voit & Mayer, Ltd.
Whisenant Ethan
LandOfFree
Solid phases for target nucleic acid detection, process for... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Solid phases for target nucleic acid detection, process for..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Solid phases for target nucleic acid detection, process for... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2554733