Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-13
2001-05-22
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100
Reexamination Certificate
active
06235479
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The field of the invention relates to methods and devices for conducting analytical or biological sample testing, such as, for example, for the purpose of identification of microorganisms or viruses in a test sample, and/or the susceptibility or resistance of the microorganisms or viruses to drugs. The invention is particularly related to the field of methods and devices for detection and analysis of nucleic acids present in a biological test sample.
2. Technical Review
Nucleic acid examination is a continually emerging area for test sample analysis including investigating genetic sequence and expression. Initially, a test sample from a human patient or other source is isolated and target nucleic acids in the sample are amplified to increase the number of copies to allow the test sample to be analyzed. The copies of target sequences are then hybridized to one or more complementary oligonucleotide probes in solution or in combination with a solid support incorporating complementary probes to form a hybridization complex. A detector probe may also be hybridized to the target sequences under some reactions. Detection and identification of the hybridization product is achieved when a signal is generated from the hybridization complex formed. By amplifying nucleic acid sequences, information about the presence or absence of microorganisms is obtained from the sample without resorting to culturing microorganisms.
An amplification method is described in U.S. Pat. No. 4,683,195 (Mullis) and U.S. Pat. No. 4,683,202 (Mullis), in which a polymerase chain reaction (PCR) utilizes DNA polymerase, complementary primer molecules and repeated cycles of thermal reactions to exponentially replicate target nucleic acid molecules. U.S. Pat. No. 5,792,607 (Backman) describes amplification methods referred to as ligase chain reactions (LCR). U.S. Pat. No. 5,744,311 (Fraiser); U.S. Pat. No. 5,648,211 (Fraiser) and U.S. Pat. No. 5,631,147 (Lohman), describe isothermal amplification systems based on strand displacement amplification (SDA). See also, Walker, et al.,
Nuc. Acids. Res.
20, 1691-1696 (1992) U.S. Pat. No. 5,437,900 (Burg) and EP 373 960 (Gingeras), describe isothermal amplification. Still other nucleic acid amplification methods are described in U.S. Pat. No. 5,399,491 (Kacian) and U.S. Pat. No. 5,409,818 (Davey). Each and all references noted above are incorporated herein by reference together with all other patents and literature references cited herein.
Hybridization techniques have been described for example, in U.S. Pat. No. 4,563,419 (Ranki) and U.S. Pat. No. 4,851,330 (Kohne) and in Dunn, et al.,
Cell
12, pp. 23-26 (1978) among many other publications. Various modifications to the hybridization reactions are known in the art including in solution hybridization or to capture probes on a solid support in one or more reaction steps.
Detection methods described in U.S. Pat. No. 4,486,539 (Kourlisky); U.S. Pat. No. 4,411,955 (Ward) U.S. Pat. No. 4,882,269 (Schneider) and U.S. Pat. No. 4,213,893 (Carrico), illustrate preparation of labeled detection probes for detecting specific nucleic acid sequences. Detectable labels have been conjugated, directly or indirectly through linker arms on either the base, sugar or phosphate moiety of one or more specific oligonucleotides. Labels known in the art include fluorochromes, radioisotopes, dyes, enzymes such as alkaline phosphatase, and luminescent or chemiluminescent molecules. The detector probes may bind to the amplified nucleic acid reaction products or amplicons. The amount of signal detected from the labeled detector probes after hybridization to amplicons reflects the presence or absence of amplicons and therefore of one or more selected target nucleic acid in the original test sample.
In general, amplifying known starting quantities of an internal control in a sample, then hybridizing with detector probes having an opposite sequence or sense to form a complex with the internal control amplicons and finally detecting signal generated provides one method to permit the qualitative and quantitative determination of target nucleic acids in a test sample.
Quantitative methods to determine the amount of target present in the test sample are described in U.S. Pat. No. 5,213,961 (Bunn); U.S. Pat. No. 5,476,774 (Wang); U.S. Pat. No. 5,705,365 (Ryder) and U.S. Pat. No. 5,457,027 (Nadeau).
Nucleic acid detection kits are commercially available and employ some of the above-referenced amplification, hybridization, labeling detection and quantitation techniques. For example, an HIV assay which detects amplified nucleic acid is described in U.S. Pat. No. 5,559,662 (Respess) and Gratzi, et al,
J. Virol. Methods
66, pp. 269-292 (1997). Kits which achieve such amplification of HIV nucleic acids include the Chiron QUANTIPLEX Branched DNA and Organon Teknika NASBA-QT.
A test kit for
M. tuberculosis
is described in U.S. Pat. No. 5,643,723 (Persing) and nucleic acids for mycobacteria testing are described in U.S. Pat. No. 5,589,585 (Mabilat); U.S. Pat. No. 5,702,317 (Mabilat) and U.S. Pat. No. 5,849,901 (Mabilat).
Nucleic acid sequence analysis can be divided into two different categories: limited detection or low detection formats, and multi-detection or high detection formats. The distinction is the number of specific data signals or responses that can be obtained from the formats.
EP 0 875 584 describes an instrument that uses a low detection formats. A single type of fluorescent label bound to a detection probe can be measured to determine the presence of complementary nucleic acids in the test sample. By sequentially using this technique, the instrument has been shown to be able to measure at least two specific targets in a test sample. Alternatively, multiple labels can be measured in a screening assay simultaneously (eg; fluorescent or luminescent labels with different emission properties). However for practical reasons, the number of target analytes that can be measured in a screening assay remains less than ten. These techniques are considered included in the category of low detection formats.
The design or selection of a test panel in a low detection format will be determined in part by customer need, by disease incidence in a particular location, by the prevalence of co-infection of microorganisms and/or viruses and environmental conditions to name but a few. While low resolution or low detection formats such as nucleic acid detection kits noted above are useful in detecting the presence of a conserved or limited number of target sequences, these tests have been less effective for obtaining higher levels of data or detail about target nucleic acids which are needed for analysis of a particular variant, its drug susceptibility or presence of a mutation.
The second category of analytical methods is a multi-detection or high detection format. These methods are capable of providing more detailed information about the target amplicons. An example of this is the reverse dot-blot technique, wherein nucleic acids are hybridized to multiple complementary probes immobilized on a matrix. After staining, the multiple sections that become stained indicate the presence of complementary target(s). Another technique provides a light-directed, spatially addressable matrix or array on which is deposited numerous oligonucleotides.
Recent advances in large scale genetic analysis utilize oligonucleotides assembled as multi-detection arrays by microfabrication techniques. Synthesis and methods of these arrays are described in U.S. Pat. No. 5,700,637 (Southern); U.S. Pat. No. 5,445,934 (Fodor); U.S. Pat. No. 5,807,522 (Brown); U.S. Pat. No. 5,202,231 (Drmanac) and U.S. Pat. No. 5,837,832 (Chee). The arrays allow hybridization reactions in which the immobilized oligonucleotides are explored by labeled probes or labeled amplicons for identification of variants or mutations such as single or multiple base substitutions. In an example of an application, multi-detection arrays for detection of human immunodeficiency viruses a
bio Merieux, Inc.
Brusca John S.
Lundgren Jeffrey S
McDonnell & Boehnen Hulbert & Berghoff
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