Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-11-12
2002-11-05
Siew, Jeffrey (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091200, C435S287100, C436S518000, C436S527000, C436S528000, C436S536000, C536S022100, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06475721
ABSTRACT:
Subject matter of the invention is a solid carrier having two or more nucleic acid analogs with different base sequences bound to predetermined sites on its surface. The invention also addresses a method for the detection of nucleic acids using a carrier of this nature.
Sample analysis has undergone rapid development in recent decades. While analytes were initially detected primarily by means of their reaction with conventional chemical reagents, and later on with enzymes, tests that utilize the immunological characteristics of the analyte have become the standard recently, especially in medical diagnostics. This is especially true in the field of infectious diseases. However, immunological procedures can basically only detect analytes with which immunologically active compounds such as antigens or antibodies play a role. These procedures have resulted in promising potential applications for many infections caused by viruses or bacteria. Genetic diseases or predispositions that are not expressed as a change in protein patterns—or only to an insufficient extent—are either difficult or impossible to detect using immunological procedures, however. Nucleic acids have therefore recently become the object of detection in many cases. The presence of certain nucleic acids can infer the presence of an infectious agent or the genetic condition of an organism. Detection procedures based on the presence of special nucleotide sequences in particular were facilitated recently when methods for the amplification of nucleic acids that are present in small numbers became available. Due to the large quantity of sequence information and the fact that two nucleotide sequences with completely different functions often differ by just one base unit, the specific detection of nucleotide sequences still poses a considerable challenge for reagents and analytical methods that are based on the detection of nucleic acid sequences. In addition, the nucleotide sequences are often not even known, but rather are determined for the first time in the nucleic acid detection method itself.
A method for the detection of nucleotide sequences of the HLA gene is described in EP-B-0 237 362 with which a clinically relevant point mutation can be detected. In this method, an oligonucleotide that is bound to a membrane and has a nucleotide sequence that is exactly complementary to one of the two nucleic acids to be differentiated is brought in contact with the sample. While certain conditions are maintained, only that nucleic acid that is exactly complementary binds to the oligonucleotide that is bound to the solid phase, and can be detected.
A method is described in Proc. Natl. Acad. Sci. USA 86, 6230-6234 (1989) in which a large number of oligonucleotides that are bound to different, predetermined sites of a nylon membrane by means of poly-dT are used for the simultaneous detection of all known allelic variants of an amplified region of a nucleic acid.
A method is described in U.S. Pat. No. 5,202,231 in which the sequence of a nucleic acid can be determined theoretically by bringing oligonucleotides having a predetermined, known sequence in contact with a sample of the unknown nucleic acids under hybridization conditions. This requires that all possible permutations of the nucleotide sequence be immobilized on known sites of a solid phase. By determining the sites to which the nucleic acids containing the sequence to be determined hybridize, it can theoretically be determined which sequences are present in the nucleic acid.
Prior art in the field of the analysis of genetic polymorphisms using “oligonucleotide arrays” is described in Nucleic Acids Research 22, 5456-5465 (1994) and Clin. Chem. 41/5, 700-6 (1995).
The main problem with the prior art is the fact that the melting temperatures of the selected sequence-specific oligonucleotides containing the nucleic acids to be sequenced or detected are different. To remedy this situation, one has to perform the complex method of selecting the length of the oligonucleotide and its base composition, and optimizing the position of the mismatches within the oligonucleotide as well as the salt concentration of the hybridization complex. In many cases, however, it is practically impossible to simultaneously distinguish closely related sequences from each other. The hybridization temperature is another critical parameter. Variations of as little as 1 to 2° C. can change the intensity or produce false-negative results. Incorrect analytical results based on the presence of point mutations have serious implications for diagnosis.
The object of this invention was, therefore, to provide an alternative method for the sequence-specific detection of nucleic acids and to provide suitable materials for this method.
This object was accomplished by providing a solid carrier having two or more nucleic acid analogs with different base sequences bound to predetermined sites on its surface. Another object of the invention is a method for the sequence-specific detection of a nucleic acid using this solid carrier.
A “solid carrier” as described by this invention refers to an object that has a surface that is so broad that specific areas can be distinguished upon it. This surface is preferably flat and larger than 5 mm
2
, and is preferably between 10 mm
2
and approx. 100 cm
2
. The carrier material is not liquid or gaseous, and preferably dissolves either not at all or incompletely in the sample fluids or reaction preparations that are used to immobilize nucleic acids to the surface. Examples of such materials are glass, plastics (e.g. polystyrene, polyamide, polyethylene, polypropylene), gold, etc. The material does not necessarily have to be completely solid itself, but rather can be made solid by the attachment of supporting materials.
The external shape of the solid carrier basically depends on the method used to detect the presence of nucleic acids on this solid carrier. It has proven to be appropriate, for instance, to select a basically planar form, e.g. a chip.
Solid carriers that are especially suitable are, therefore, polystyrene chips that are from 1 to 5 mm thick and have a surface area of from 1 to 5 cm
2
, for instance. Polyamide membranes that are 4×2.5 cm
2
in size have proven to be especially well-suited for use with this invention. Two or more nucleic acid analogs having different base sequences are bound to different sites of the surface of this carrier. These sites or regions preferably do not overlap with each other. They are preferably separated from each other by regions on the surface to which no nucleic acid analogs are bound. The sites to which the nucleic acid analogs are bound are referred to as “binding regions” below. The binding regions can have different shapes. These shapes are basically determined by the method of manufacturing the solid carrier or by the method used to determine the nucleic acid analogs in the binding regions. The minimum size of the binding regions is basically determined by the instrument with which the event—the binding of a nucleic acid to nucleic acid analogs of a region—is detected. Instruments are already available that can detect binding to regions that are approx. 1 mm in size. The upper limit of the size of the binding regions is determined by cost effectiveness and handling considerations.
The size of the binding regions is also basically determined by the methods used to apply the nucleic acid analogs to the surface. Such methods will be described later.
The number of binding regions on the solid carrier depends on the intended use of the solid carrier. In the simplest case, just two binding regions are needed to detect a certain point mutation. In this case, a binding region contains nucleic acid analogs that have a base in the position at which the point mutation is to be detected. This base is complementary to the base in the position of the normal sequence. The other binding region, on the other hand, contains a nucleic acid analog that has a base in the corresponding position that is complementary to the base of the mutate
Geiger Albert
Kleiber Jörg
Lester Ane-Ullerup
Ørum Henrik
Arent Fox Kintner & Plotkin & Kahn, PLLC
Boston Probes, Inc.
Siew Jeffrey
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