Probes and methods for polynucleotide detection

Details Probes and methods for polynucleotide detection Probes and methods for polynucleotide detection

1998-06-16

2001-09-04

Marschel, Ardin H. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S022100, C536S025300

Reexamination Certificate

active

06284462

ABSTRACT:

This application is a 371 filing of PCT/JP97/03438, filed Sep. 26, 1997.
TECHNICAL FIELD
This invention relates to techniques for detection probes and method of detection for detecting a specimen that has specified polynucleotide base (DNA, RNA or the like ), by mixing detection probes, which are labeled with fluorescent dyes and can be bound to the specimen, into a specimen sample containing a specimen and measuring the fluorescence emitted by the specimen sample.
BACKGROUND ART
For methods for detecting and quantifying DNAs or RNAs having specified base sequences present in samples, methods utilizing “detection probes” that specifically hybridize to DNAs or RNAs, which are the subject of detection, are widely in use. Oligonucleotide nucleic acids having base sequences complementary to parts of the base sequences for DNAs or RNAs (target nucleic acids), which are the subject of detection, are frequently used as detection probes.
In these methods, after hybrids between detection probes and target nucleic acids have been formed, any changes resulting from the formation of the hybrids are detected, thereby confirming that the target nucleic acids to be the subject of detection are contained in samples and quantifying their contents. For example, a detection probe is labeled with a fluorescent dye. This fluorescent labeled detection probe(fluorescent labeled oligonucleotide nucleic acid) is added to a sample. If a target nucleic acid is present in the sample, the fluorescent labeled detection probe binds to the target nucleic acid to form a hybrid. Then, a manipulation is performed to separate the fluorescent labeled detection probe that does not bind from the fluorescent labeled detection probe that has been hybridized to the target nucleic acid in the sample, thereby removing the fluorescent labeled detection probe that does not bind in the sample. Here, if the fluorescence intensity of the sample is measured, it will enable the amount of the target nucleic acid in the sample to be quantified.
The above-described method needs a manipulation for removing the detection probe that does not bind to the target nucleic acid after the detection probe has been added to the sample. Because such a manipulation for separation is complicated in practice, a variety of assays that do not require manipulations for separating non-bound probes from bound probes after addition of probes have been attempted(homogeneous assays).
One of the homogeneous assays is a method that utilizes resonance energy transfer occurring between two kinds of fluorescent molecules. Generally, when two kinds of fluorescent molecules are within a distance of about 70-80 angstroms, interaction between the fluorescent molecules occurs(resonance energy transfer) and thus their fluorescence spectrum or fluorescence decay curve changes. In a fluorescence spectrum, the fluorescence intensity resulting from a donor (in general between the two kinds of fluorescent molecules, the molecule whose absorption spectrum is on the shorter wavelength side) decreases, whereas the fluorescence intensity resulting from an acceptor (between the two kinds of fluorescent molecules, the molecule whose absorption is on the longer wavelength side) increases. Also, with respect to changes in the fluorescence decay curve after pulse-excitation, decay for the donor accelerates, whereas decay for the acceptor delays.
Some attempts are made to utilize this resonance energy transfer between fluorescent molecules in the homogeneous assays for nucleic acids. Specifically, two kinds of fluorescent labeled detection probes(those individually labeled with different kinds of fluorescent dye molecules) are provided and these hybridize to a target nucleic acid adjacently with each other. The energy transfer occurs to cause changes in the fluorescence spectrum, because the two kinds of fluorescent dyes are within a close distance in a hybrid. Namely, the fluorescence spectrum changes as a result of the formation of the hybrid comprising the two kinds of fluorescent labeled detection probes and the target nucleic acid, which will enable the detection of the target nucleic acid by measuring the change in the fluorescence spectrum (Cardullo, R. A., et al. (1988) Proc. Natl. Acad. Sci. USA. 85, 8790-8794, EP0070685). U.S. Pat. No. 4,996,143 discloses oligonucleotide probes labeled with fluorescence dyes suited for methods of detecting target nucleic acids based on measurement of changes in fluorescence spectra.
Accordingly, the method to measure any change in a fluorescence spectrum caused by the energy transfer is useful for the homogeneous assays of nucleic acids. However, if the amount of detection probes in a sample (the number of their molecules) exceeds that of a target nucleic acid (the number of its molecules), practically it becomes very difficult to use the above-described method for detecting a target nucleic acid based on changes in a fluorescence spectrum. Namely, the fluorescence spectrum to be measured is the sum of a fluorescence spectrum resulting from a small number of fluorescent dye molecules that has undergone the energy transfer and a fluorescence spectrum resulting from a large number of fluorescent dye molecules that has not undergone the energy transfer. Thus, the fluorescence spectrum resulting from the small number of fluorescent dye molecules that has undergone the energy transfer is buried in the fluorescence spectrum resulting from the large number of fluorescent dye molecules that has not undergone the energy transfer, which makes it practically impossible to detect any changes in the fluorescence spectrum caused by the energy transfer.
It quite often happens in the actual measurement of biological samples that the amounts of detection probes exceed those of nucleic acids (target nucleic acids), which are the subject of detection. One such example is that the amount of a target nucleic acid in a sample is unknown. Also, the concentrations of detection probes in samples can not be lowered below certain levels, because sensitivity in measurement depends on the fluorescence intensities of the samples. Thus, when the amounts of the target nucleic acids are very small (low concentrations), the amounts of the detection probes exceed those of the target nucleic acids.
Accordingly, there is a need for the method that will allow the highly accurate detection of a nucleic acid, which is the subject of detection, even under the conditions where detection probes are present in excess relative to a nucleic acid, which is the subject of detection.
In general, among methods for detecting and measuring energy transfer between fluorescent molecules, there are a method to measure any changes in a fluorescence spectrum and a method to measure any changes in the decay curve of fluorescence intensities after pulse excitation (time-resolution method). Where fluorescent molecules that have undergone the energy transfer and the same kind of fluorescent molecules that have not undergone the energy transfer coexist, it often happens that the method to measure a fluorescence decay curve (time-resolved measurement ) is more advantageous than the method to measure changes in a fluorescence spectrum Morrison, L. E. (1998) Anal.Biochem. 174 101-120; Japanese Laid-Open Patent Application Hei 7-229835). The fluorescence decay of an acceptor delays due to the energy transfer. Morrison disclosed that when the delay in decay is sufficiently large, it is possible to selectively measure the fluorescence resulting from the acceptor excited by the energy transfer by measuring fluorescence intensities in a time zone after the fluorescence decay resulting from the acceptor directly excited is substantially complete. In Japanese Laid-Open Patent Application Hei 7-229835, there is provided a method for calibrating errors in detection that are caused by mixing of fluorescence resulting from a donor into the fluorescence wavelength region of an acceptor, said method measuring fluorescence decay in the fluorescence wavelength of the donor in addition to fluorescence decay in th

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