Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-06
2001-02-27
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06194148
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for microscopically detecting a nucleic acid molecule hybridized with a probe.
2. Description of the Related Art
In detecting a nucleic acid having a specific sequence, use is made of a method using a probe labeled with a radioisotope or fluorescent substance, a method of measuring the activity of an enzyme immobilized onto a probe, and a method for enzymatically amplifying a specific sequence, such as in PCR (polymerase chain reaction) (see EPO192168A2, WO91/14788, U.S. Pat. No. 4,734,363 etc.). These methods include the step of amplifying signals by certain means and raise the problem that this amplification step or a preliminary step therefor is so cumbersome that a longer time is required.
In addition, in cases where a probe labeled with a radioisotope, fluorescent substance or enzyme is used, while the amplification of the desired signals is accomplished through the labeled prove which has been bound to the target sequence, noises are also amplified through the probe which has been non-specifically bonded to a solid phase carrier having a sample and the probe immobilized thereon, thereby bringing about the problem of adverse influence on detection accuracy. To prevent this problem, it is necessary to take measures such as vigorous washing after hybridization, inclusion of other DNA at the time of hybridization, etc. However, even if such measures are taken, it is not possible to prevent nonspecific binding completely, and if the concentration of the target nucleic acid molecule is low, higher noises are caused by nonspecifically bound nucleic acid molecules, which may result in failing to detect the signals from specifically bound nucleic acids. Further, because these additional procedures make the inherently complicated experimental operation further cumbersome, there is the drawback of increases in the labor and time required for the experiment. In addition, probe-labeling operation itself also requires cumbersome procedures. Further, if a radioisotope is used, there are potential problems that a place for conducting the experiment is limited, and qualification as the experimenter is required, etc.
On the other hand, if the PCR method is used for detecting DNA, it is disadvantageous that the step of enzymatically amplifying DNA needs a longer time, and further steps of fractionating DNA by gel electrophoresis etc. is required after the amplification step. Further, a spectrophotometer, fluorescence spectrophotometer or fluorescence microscope is used for detection of signals of labeled nucleic acid molecules. In these detection systems, the target nucleic acid molecule hybridized with the probe is detected by detecting the activity of the enzyme or the fluorescence of the fluorescent substance with which the probe has been labeled. Accordingly, these detection systems suffer from the problem that the specific binding between the nucleic acid probe and the target nucleic acid molecule cannot be distinguished from the nonspecific binding of the nucleic acid probe to other nucleic acids than the target nucleic acid molecule or to the solid carrier surface.
As described above, the conventional methods of detecting nucleic acid make use of a labeled nucleic acid probe, so the step of removing the unbound nucleic acid probe by washing is required to eliminate background noises from data at the time of measurement. Further, the subject to be detected by labeling is the presence of the labeled substance itself, but not the binding itself of the probe to the target nucleic acid molecule. Therefore, there is no means of distinguishing noises caused by nonspecific binding from the signal of specific binding. As a result, if the concentration of the target nucleic acid molecule is low, noises caused by nonspecific binding are higher than the signal from specific binding, which may lead to the erroneous conclusion that the target nucleic acid molecule is not present in the sample. On the other hand, if the nonspecifically bound nucleic acid probe remains due to insufficient washing, the conclusion that the target molecule is present in the sample may be derived although the target nucleic acid is not actually present. Furthermore, because this nonspecific binding cannot be eliminated completely, it cannot be determined whether the signals detected are due to an increase in nonspecific binding resulting from insufficient washing etc. or due to specific binding when the signals are slightly higher than noises caused by nonspecific binding. Thus, such signals are considered as pseudo-positive data and it cannot be determined, in some cases, whether these data are inherently positive or negative.
SUMMARY OF THE INVENTION
Accordingly, an object of the present invention is to clearly distinguish, without confusion, specific binding of a probe to a target nucleic acid from a nonspecific one by means of directly detecting a target nucleic acid molecule hybridized with the probe without using any label. Another object of the present invention is to easily detect a nucleic acid molecule without removing unbound components by washing etc. A further object of the present invention is to detect a target nucleic acid molecule accurately in a short time.
These objects can be attained by a method for detecting a target nucleic acid molecule hybridized with a nucleic acid probe, comprising the steps of:
(a) hybridizing a target nucleic acid molecule having a specific sequence to be detected in a sample with a nucleic acid probe having a sequence complementary to the sequence of the nucleic acid molecule, the nucleic acid probe being selected such that the nucleic acid molecule and the probe are partially hybridized to represent a linear conformation in a hybridized region and to represent, in an non-hybridized region, a unique structure inherent to its nucleotide sequence;
(b) measuring the conformation, structure and length of the nucleic acid molecule obtained in step (a); and
(c) on the basis of the measurement data obtained in the step (b), detecting the nucleic acid containing both of the linear conformation and the unique structure, thereby detecting the nucleic acid molecule hybridized with the probe.
In the present invention, the nucleic acid probe may be longer or shorter than or equal to the target nucleic acid molecule to be detected, insofar as a hybrid nucleic acid having a linear double-stranded portion and a folded single-stranded portion is formed upon hybridization of the probe with the target nucleic acid molecule. Further, two or more probes hybridizing with different regions in the target nucleic acid molecule can also be used.
The term “linear” used in the present invention does not mean that a nucleic acid molecule is straight shape but means that a nucleic acid is extending without folding. In general, the hybridized or renatured nucleic acid molecule has a linear conformation in nature.
The term “unique structure inherent to its nucleotide sequence” used herein is based on the finding that single-stranded nucleic acid has an “unnatural conformation”, i.e., a unique structure depending on its nucleotide sequence. Such a unique structure includes, but is not limited to, at least one horn-like portion folded at least one portion, a hook-like portion, a site that seems looped when viewed in at least one direction, or a globular-like portion such as a three-dimensional polyhedral, globular or amorphous mass.
Normally, the hybridized double-stranded nucleic acid is “linear” as described above and does not show such a unique structure as that of single-stranded nucleic acid. However, it is not almost straight or absolutely straight but is gradually distorted as a whole. On the other hand, the single-stranded portion having a “unique structure inherent to its nucleotide sequence” may have an almost straight or absolutely straight portion.
Once the nucleotide sequence is determined, the conformation of its high-order structure can be defined according to its sequence and length. Accordingly
Hori Kunio
Okada Takao
Takahashi Takeo
Frishauf, Holtz Goodman, Langer & Chick, P.C.
Olympus Optical Co,. Ltd.
Whisenant Ethan
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