Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-16
2003-01-07
Whisenant, Ethan C. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06503714
ABSTRACT:
Subject matter of the invention is a method of detecting a substance to be analyzed in a sample using a probe which contains nucleobases.
The detection of low-concentration substances to be analyzed in samples has become very important in clinical testing. As a result of numerous developments, the detection limit for substances to be analyzed has been lowered considerably compared with earlier tests. This development has had a particularly strong impact in the field of nucleic acid detection. Many of the detection systems used today, however, involve complex working steps or use reagents that are very complex to manufacture. These systems have many disadvantages.
In U.S. Pat. No. 4,683,202, a method for detecting nucleic acids is described that is based on the amplification of segments of an original nucleic acid. This method has become known as the polymerase chain reaction. A disadvantage of this method, however, is that the results are difficult to quantify.
Results are easier to quantify when nucleic acid probes are used with which stoichiometric hybridization takes place. Unfortunately these methods are insensitive.
In a further development of the traditional hybridization test, it was therefore proposed that a number of identical nucleotide sequences be bound with each probe. The hybrid consisting of analyte nucleic acid and probe was detected by hybridizing a number of secondary probes that were complementary to these identical nucleotide sequences. A number of documents that describe the prior art discuss the most favorable arrangement of the many identical nucleotide sequences in the probe or in a set consisting of probes that are capable of hybridizing—and, therefore, aggregating—with each other (e.g., U.S. Pat. No. 5,424,188; EP-B-0 248 896 B1; U.S. Pat. No. 5,424,413; 5,437,977). It is stated in U.S. Pat. No. 5,424,188, for instance, that the identical nucleotide sequences can be attached to each other in linear fashion. The chain of identical nucleotide sequences produced can also be formed into a closed ring. It is stated in U.S. Pat. No. 5,124,246 that the identical nucleotide sequences can be bound to each other by means of a branched structure. A distinction is made in this case between fork-like branchings and comb-like branchings. A number of potential technical embodiments have been described for the branchings (Nucleic Acids Research 16/11, 4937-4965, Gene 61, 253-264, Nucleic Acids Research 17/17, 6959-6967, Clinical Chemistry 35/8, 1571-1575 (1989), Bioorganic and Medicinal Chemistry Letters 4/8, 1011-1018 (1994), Nucleic Acids Research Symposium 24, 197-200 (1991), and Clin. Chem. 39/4, 725-726 (1993)). In WO 95/01365, a branching method is also described in which the lateral arms are bound to a special backbone by means of a reaction between phosphorothioates and haloacyl groups. Methods in which the probe has a number of identical nucleotide sequences for hybridizing with secondary probes can often lead to increased calibration curve sensitivity. The probes required for this, however, are 1) difficult to manufacture, and 2) the labelled probes do not stoichiometrically saturate the nucleic acid sequences on the probes. The test sensitivity achieved is therefore much less than that of the PCR method (AIDS 7/suppl., 11-14 (1993); J. Med. Virol. 43, 262-268 (1994); J. Infect. Dis. 170, 1172-1179 (1994); AIDS Res. & Human Retrovir. 11/3, 353-361 (1995)).
The one feature that all of these concepts have in common is that at least two different types of probes—one of which is present in great numbers—are aggregated into one detectable amplification unit based on the principle of hybridization of single-stranded overhangs.
Object of the invention was, therefore, to improve the prior art and, in particular, to develop a sensitive method of detection that can be quantified directly.
Subject matter of the invention is therefore a method for detecting a substance to be analyzed in a sample by contacting the sample with a probe which contains nucleobases and two or more non-nucleosidic, label-attracting groups under conditions in which the substance to be analyzed binds indirectly or directly to the probe and detecting the binding product.
A substance to be analyzed according to the invention can be any ingredient in a sample. Preferably, however, the substance to be analyzed is a molecule that can be detected immunologically or by means of base pairing. Immunologically detectable substances to be analyzed are, for instance, antibodies, antigens or haptens. Nucleotides are substances to be analyzed that can be detected by means of base pairing. They include ribonucleic acids and deoxyribonucleic acids. These types of substances to be analyzed can be detected in practically any sample using the proposed method. In many cases, however, it is preferable to pretreat the sample so that the substance to be analyzed is present in a form that facilitates detection. This includes releasing the substance to be analyzed from compartments, e.g., cells, in which the analyte was originally enclosed. The substance to be analyzed can itself be a compartment, however, if it has detectable components on its surface, such as surface antigens. In addition, the actual substance to be analyzed can be amplified before the method provided by this invention is carried out, e.g., using the PCR, but preferably only to the degree that the amplification can still be quantified. Blood, urine, sputum or swabs have proven to be suitable fluids from which a sample suitable for detection can be produced. Another possible preparation step is to liquify viscous samples or to partially purify the analyte, e.g., by immobilizing it on a solid phase. The analyte can be present in a liquid, e.g., in dissolved or suspended form. The analyte can also be bound to a solid phase, however. Suitable solid phases include latex particles, magnetic particles, or vessel walls.
A probe according to the present invention is a molecule that has a first nucleic acid-type portion, and a second portion that is analyte-specific or that promotes contact with the analyte. The nucleic acid-type portion contains nucleobases on a backbone. Nucleobases include the naturally occurring bases A, G, C, T, and U, or any bases derived therefrom. A potential backbone is the natural sugar phosphate structure. Molecules that have a polyamide backbone were also described recently (e.g., in WO 91/20702). The probe can bind the analyte directly or indirectly by means of the analyte-specific portion. In cases of direct binding, the nature of the analyte-specific portion can depend on the type of substance to be analyzed. If the substance to be analyzed is immunologically active, binding can take place with the partner that is immunologically complementary to the analyte. To detect an antigen, for instance, a probe can therefore be used that contains an antibody to this antigen that is bound covalently or non-covalently to the nucleic acid-type portion. If the substance to be analyzed is a nucleic acid, the probe contains a nucleobase sequence that is complementary to one part of the base sequence of the analyte. An indirect binding of the probe to the analyte can be achieved by directing the analyte-specific portion against a part of a promotor probe that can bind with the analyte. The promoter probe therefore preferably contains an analyte-specific portion and a portion that is capable of binding with the analyte-specific portion. In this case it is preferable for the probe and the promoter probe to bind with each other by means of base pairing. In this case, a promoter probe is preferably selected that has a portion that is complementary to the analyte and a portion that is complementary to the probe. The advantage of this method is that a number of relatively simply constructed promoter probes be used that correspond to the number of analytes to be detected in order to detect various analytes, but only requires one relatively complex probe.
The nucleic acid-type portion of the probe is designed in such a way that it cannot form hybrids
Lassonczyk Gerhard
Seidel Christoph
Weindel Kurt
Arent Fox Kintner & Plotkin & Kahn, PLLC
Roche Diagnostics GmbH
Whisenant Ethan C.
LandOfFree
Method of detecting a substance to be analyzed does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of detecting a substance to be analyzed, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of detecting a substance to be analyzed will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3063957