Assay of nucleic acid by fluorescence polarization technique...

Details Assay of nucleic acid by fluorescence polarization technique... Assay of nucleic acid by fluorescence polarization technique...

1998-08-03

2001-12-11

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S022100, C536S024300, C536S025320

Reexamination Certificate

active

06329142

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method for rapidly assaying a nucleic acid in a sample amplified by gene amplification by a fluorescence polarization technique. More particularly, it relates to a method for rapidly detecting a Verotoxin-producing microorganism by making use of this assay.
BACKGROUND OF THE INVENTION
The necessity of assaying nucleic acids, the entity of genes, has been increasing. Methods utilizing radioisotopic labels or enzymatic labels have been studied on the model of conventional immunoassay, and some of them have been put into practical use. Most of these conventional methods for nucleic acid assay are based on a heterogeneous system. That is, a sample containing a nucleic acid to be assayed is mixed with a reagent to cause a reaction, separating the reacted substance or reagent (bound form) and unreacted substance or reagent (free form) (called B/F separation), and measuring signals sent from the label. The B/F separation is carried out by the use of a DNA reagent or magnetic fine particles or by electrophoresis. Any of these known B/F separation techniques require complicated or time-consuming operations.
A fluorescence polarization technique is known to be applicable to homogeneous assay systems which do not involve the B/F separation. While this technique has been known as a simple, easy and rapid method for determining a drug, etc. in a sample, it is deemed applicable to the assay of nucleic acids as well (see Unexamined Published Japanese Patent Application No. 5-123196).
In carrying out the assay of nucleic acid by the fluorescence polarization technique, a nucleic acid containing a base sequence complementary to the nucleic acid to be detected is labeled with a fluorescent substance to prepare a fluorescence-labeled reagent (also called a labeled probe). A single-stranded nucleic acid is usually used as a reagent.
An example of nucleic acid assay by a fluorescence polarization technique is as follows. A fluorescence-labeled reagent is added to a sample for assay. If the sample contains a nucleic acid having a target base sequence, the site of the nucleic acid having that base sequence is engaged and bound to the complementary reagent. This reaction is called hybridization. The nucleic acid in the sample should previously be denatured by heat treatment or treatment with a chemical so as to have a single-stranded structure. Upon hybridization between the fluorescence-labeled reagent and the target nucleic acid, the apparent molecular weight of the reagent increases. In general, movement of molecules in a solution becomes slower as the molecular weight increases. Therefore, the degree of fluorescence polarization after hybridization is higher than before hybridization because of the increase of the fluorescence-labeled reagent in apparent molecular weight. With the amount of the fluorescence-labeled reagent being fixed, the change in degree of fluorescence polarization is proportional to the amount of the nucleic acid in the sample. Therefore, the amount of the target nucleic acid in the sample can be measured from the change in degree of fluorescence polarization by the reaction.
The degree of fluorescence polarization is usually measured by setting a polarizer on both the exciting side and the fluorescence side, rotating the polarizer on the fluorescence side, and measuring fluorescence whose plane of polarization is parallel to the plane of polarization of the exciting light and fluorescence whose plane of polarization is perpendicular to the plane of polarization of the exciting light. Therefore, one measurement is completed within such a short time as 1 minute.
As stated above, a fluorescence polarization technique does not require B/F separation and is expected to establish a rapid and easy method for assaying nucleic acids. However, the detection sensitivity of this method is not so high as expected because the technique essentially relies for its sensitivity on the detection sensitivity of the fluorescence-labeled substance. Further, in many cases in which a sample taken from a patient or a food is examined, the amount of the nucleic acid, if any, is so small that the sensitivity of the fluorescence polarization technique is not enough.
It may be easily assumed that the sensitivity could be improved by amplifying the genes (nucleic acids) of the target microorganism by gene amplification such as the PCR method as taught, e.g., in Erlich, H. A., Gelfand, D. H. and Saiki, R. K.,
Nature
, Vol. 331, pp. 461-462, “Specific DNA Amplification” (1988). It has also been proposed to use a fluorescence-labeled oligoDNA as a primer for amplification of a gene nucleic acid so as to increase the degree of fluorescence polarization with the progress of amplification as reported in Tamiya, E. and Karube, I.,
New Functionality Materials
B, pp. 99-104 (1993).
As mentioned above, it is expected that the amount of a nucleic acid, such as deoxyribonucleic acid (DNA), is increased by gene amplification and the amplification product can be detected by the fluorescence polarization technique.
However, in experimentation on DNA in which DNA is amplified by a usual polymerase chain reaction (PCR) technique (see Erlich, H. A., Gelfand, D. H. and Saiki, R. K.,
Nature
, Vol. 331, pp. 461-462, “Specific DNA Amplification” (1988)), and the amplified DNA is assayed as such by the fluorescence polarization technique, the results obtained have often turned out still insufficient in detection sensitivity or poor in reproducibility.
SUMMARY OF THE INVENTION
An object of the present invention is to overcome the disadvantage of the conventional techniques and to provide a method for assaying a nucleic acid in a sample having been amplified by gene amplification accurately, rapidly, and with good reproducibility by making use of fluorescence polarization.
Another object of the present invention is to provide a method for rapidly detecting a Verotoxin-producing microorganism, such as
E. coli
O-157, by using the above method.
The inventors of the present invention considered that such a disadvantage of the fluorescence polarization technique applied to a nucleic acid amplification product as obtained by usual PCR is ascribable to the following. An amplification product of a nucleic acid as obtained by usual PCR is substantially a complete double-stranded nucleic acid, whereas a fluorescence-labeled reagent used in fluorescence polarization is, in principle, has a relatively short single-stranded structure. When the longer double strand nucleic acid in a sample is denatured into a single-stranded nucleic acid by, for example, heat treatment, and hybridization is tried between the fluorescence-labeled reagent and a target single-stranded nucleic acid that has a base sequence complementary to the labeled reagent, the other single-stranded nucleic acid complementary to the target s—s nucleic acid competes with the fluorescence-labeled reagent, making the hybridization difficult in terms of energy. Thus, a mere combination of a fluorescence polarization technique and a known technique of gene amplification, such as a standard PCR, fails to establish a nucleic acid assay system having good reproducibility and high sensitivity.
As a result of extensive investigations, the inventors have found that the above objects are accomplished by carrying out gene amplification by asymmetric gene amplification thereby to selectively amplify the target single-stranded nucleic acid that has a base sequence complementary to a fluorescence-labeled reagent. According to this method, since the target single-stranded nucleic acid predominates over the other single-stranded nucleic acid, the efficiency in hybridization between the fluorescence-labeled reagent and the target single-stranded nucleic acid is obviously improved.
They have also found that the above objects are accomplished by amplifying the nucleic acid by gene amplification, denaturing the amplified nucleic acid by heat treatment, etc. into a single-stranded nucleic acid, and annealing them with the primers used in gene

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