Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-01-03
2001-10-30
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C422S050000, C422S068100
Reexamination Certificate
active
06309823
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides arrays of oligonucleotide probes immobilized in microfabricated patterns on chips for analyzing biotransformation genes, such as cytochromes P450.
2. Description of Related Art
Virtually all substances introduced into the human body (xenobiotics) as well as most endogenous compounds (endobiotics) undergo some form of biotransformation in order to be eliminated from the body. Many enzymes contribute to the phase I and phase II metabolic pathways responsible for this bioprocessing. Phase I enzymes include reductases, oxidases and hydrolases. Among the phase I enzymes are the cytochromes P450, a superfamily of hemoproteins involved in the oxidative metabolism of steroids, fatty adds, prostaglandins, leukotrienes, biogenic amines, pheromones, plant metabolites and chemical carcinogens as well as a large number of important drugs (Heim & Meyer,
Genomics
14, 49-58 (1992)). Phase II enzymes are primarily transferases responsible for transferring glucuronic acid, sulfate or glutathione to compounds already processed by phase I enzymes (Gonzales & Idle,
Clin. Pharmacokinet.
26, 59-70 (1994)). Phase II enzymes include epoxide hydrolase, catalase, glutathione peroxidase, superoxide dismutase and glutathione S-transferase.
Many drugs are metabolized by biotransformation enzymes. For some drugs, metabolism occurs after the drug has exerted its desired effect, and result in detoxification of the drug and elimination of the drug from the body. Similarly, the biotransformation enzymes also have roles in detoxifying harmful environmental compounds. For other drugs, metabolism is required to convert the drug to an active state before the drug can exert its desired effect.
Genetic polymorphisms of cytochromes P450 and other biotransformation enzymes result in phenotypically-distinct subpopulations that differ in their ability to perform biotransformations of particular drugs and other chemical compounds. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to most human may cause intolerable side-effects in an individual suffering from a defect in an enzyme required for detoxification of the drug. Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of lack of a enzyme required for conversion of the drug to a metabolically active form. Further, individuals lacking a biotransformation enzyme are often susceptible to cancers from environmental chemicals due to inability to detoxify the chemicals. Eichelbaum et al.,
Toxicology Letters
64/65, 155-122 (1992). Accordingly, it is important to identify individuals who are deficient in a particular P450 enzyme, so that drugs known or suspected of being metabolized by the enzyme are not used, or used only with special precautions (e.g., reduced dosage, close monitoring) in such individuals. Identification of such individuals is also important so that such individuals can be subjected to regular monitoring for the onset of cancers.
Existing methods of identifying deficiencies are not entirely satisfactory. Patient metabolic profiles are currently assessed with a bioassay after a probe drug administration. For example, a poor drug metabolizer with a CYP2D6 defect is identified by administering one of the probe drugs, debrisoquine, sparteine or dextromethorphan, then testing urine for the ratio of unmodified to modified drug. Poor metabolizers (PM) exhibit physiologic accumulation of unmodified drug and have a high metabolic ratio of probe drug to metabolite. This bioassay has a number of limitations: lack of patient cooperation, adverse reactions to probe drugs, and inaccuracy due to coadministration of other pharmacological agents or disease effects. Genetic assays by RFLP (restriction fragment length polymorphism), ASO PCR (allele specific oligonucleotide hybridization to PCR products or PCR using mutant/wildtype specific oligo primers), SSCP (single stranded conformation polymorphism) and TGGE/DGGE (temperature or denaturing gradient gel electrophoresis), MDE (mutation detection electrophoresis) are time-consuming, technically demanding and limited in the number of gene mutation sites that can be tested at one time.
The difficulties inherent in previous methods are overcome by the use of DNA chips to analyze mutations in biotransformation genes. The development of VLSIPS™ technology has provided methods for making very large arrays of oligonucleotide probes in very small areas. See U.S. Pat. No. 5,143,854, WO 90/15070 and WO 92/10092, each of which is incorporated herein by reference. U.S. Ser. No. 08/082,937, filed Jun. 25, 1993, describes methods for making arrays of oligonucleotide probes that can be used to provide the complete sequence of a target nucleic acid and to detect the presence of a nucleic acid containing a specific nucleotide sequence. Others have also proposed the use of large numbers of oligonucleotide probes to provide the complete nucleic acid sequence of a target nucleic acid but failed to provide an enabling method for using arrays of immobilized probes for this purpose. See U.S. Pat. No. 5,202,231, U.S. Pat. No. 5,002,867 and WO 93/17126.
Microfabricated arrays of large numbers of oligonucleotide probes, called “DNA chips” offer great promise for a wide variety of applications. The present application describes the use of such chips for inter alia analysis of the biotransformation genes, such as cytochromes P450.
SUMMARY OF THE INVENTION
The invention provides arrays of probes immobilized on a solid support for analyzing biotransformation genes. In a first embodiment, the invention provides a tiling strategy employing an array of immobilized oligonucleotide probes comprising at least two sets of probes. A first probe set comprises a plurality of probes, each probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of a reference sequence from a biotransformation gene, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence. A second probe set comprises a corresponding probe for each probe in the first probe set, the corresponding probe in the second probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets. The probes in the first probe set have at least two interrogation positions corresponding to two contiguous nucleotides in the reference sequence. One interrogation position corresponds to one of the contiguous nucleotides, and the other interrogation position to the other. In this, and other forms of array, biotransformation genes of particular interest for analysis include cytochromes P450, particularly 2D6 and 2C19, N-acetyl transferase II, glucose 6-phosphate dehydrogenase, pseudocholinesterase, catechol-O-methyl transferase, and dihydropyridine dehydrogenase.
In a second embodiment, the invention provides a tiling strategy employing an array comprising four probe sets. A first probe set comprises a plurality of probes, each probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of a reference sequence from a biotransformation gene, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence. Second, third and fourth probe sets each comprise a corresponding probe for each probe in the first probe set. The probes in the second, third and fourth probe sets are identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the at least one interrogation position, except that the at le
Chee Mark
Cronin Maureen T.
Fodor Stephen P. A.
Huang Xiaohua C.
Hubbell Earl A.
Affymetrix Inc.
Marschel Ardin H.
Townsend and Townsend / and Crew LLP
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