Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2001-05-25
2003-08-19
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Vector, per se
C435S006120, C435S091100, C435S091400, C435S091500, C536S023100
Reexamination Certificate
active
06607911
ABSTRACT:
BACKGROUND OF THE INVENTION
Nucleic acids encompass both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA, present in all nucleated cells, carries the information needed to direct the synthesis of every protein in the body. A single alteration in the correct sequence of the four DNA bases (adenine, thymidine, guanine, and cytosine) may result in a defective protein. Depending upon the protein and the affected organism, the defect may range from inconsequential to life-threatening, or may be of intermediate severity. Diseases as diverse as cystic fibrosis, some types of cancer, sickle cell anemia, and atherosclerosis are known to result from specific genetic alterations.
RNA, the intermediary between DNA and protein, is the product of transcription of a DNA template. RNA assays are being performed with increasing frequency in research and clinical laboratories. This is due at least in part to the prevalence of RNA viruses such as the human immunodeficiency virus (HIV) that causes AIDS and the hepatitis C virus (HCV), and the development of drugs used in treating infections with RNA viruses. Precise testing for the presence of specific nucleic acid sequences for identification or monitoring of disease is important, and constructs comprising nucleic acids with known sequences are necessary for validation, calibration, and standardization of those tests.
Nucleic acid assays are routinely performed, either manually or by automated instrumentation, in numerous reference and clinical laboratories. A nucleic acid assay may be performed to detect the presence of foreign DNA or RNA, which may indicate infection with a foreign organism. For example, a variety of molecular assays are used to establish the presence and identity of nucleic acids from the human immunodeficiency virus-1 (HIV-1), Chlamydia, and other organisms causing sexually transmitted diseases. An individual's DNA may also be analyzed to detect, treat, and in some cases prevent genetic disease. Genotype determination of genes for Factor-V Leiden, hereditary hemochromatosis, lipoprotein lipase mutations, and cystic fibrosis have important implications for health management. The Human Genome Project holds the promise of many more examples of medically efficacious genetic diagnostic determinations. The recent discovery of the breast cancer associated gene (BRCA-1) has highlighted both the importance of screening individuals for predisposition to a disease, and also the attendant need for accurate, precise, reproducible, and controlled nucleic acid assays.
Nucleic acid testing of a patient derived specimen is a multi-step process. Failure of any step in the process leads to inaccurate clinical information with potentially serious outcome for the patient. The clinical nucleic acid testing protocol includes amplification of one or more DNA segments, and detection of product by any of a number of techniques including binding and detection of labeled probe, and/or restriction enzyme digestion and/or electrophoresis. The test may fail to give the correct result due to interfering substances, unsuitable reaction conditions, reagent problems or detection system failure. The test may be functioning in most aspects but have lost its sensitivity to detect specific mutations or to detect low levels of a given nucleic acid sequence. Some tests experience interference from unexpected polymorphisms or rare mutations and subsequently yield erroneous results. All of these errors may be detected by testing suitable known reference materials in parallel with the patient specimen. Detection of the expected signal from appropriate quality controls validates the testing process.
Current mutation detection technologies fall into two broad categories. The first group includes mutation-scanning technologies; such as single-strand conformational polymorphism (SSCP), modified double gradient gel electrophoresis (DG-DGGE), heteroduplex analysis (HET), various cleavage assays, and direct sequencing. These procedures are generally too difficult and time-consuming for most diagnostic laboratories. In the second group are methodologies more amenable to high throughput diagnostic testing. These include multiplex allele-specific diagnostic assay (MASDA), amplification refractory mutation system (ARMS), PCR followed by an oligonucleotide ligation assay and sequence-coded separation (PCR/OLA/SCS), PCR-mediated site-directed mutagenesis (PSM), and various versions of forward and reverse allele-specific oligonucleotide (ASO) dot blots. All of these assays begin with amplification of 200 to 500 base pair fragments from genomic DNA. Appropriate reference material for these assays should contain one or more of these genomic nucleic acid fragments.
In the field of molecular pathology and genetic testing, a quality control sample includes a reference DNA or reference RNA of known quantity and quality to evaluate the reliability of all steps of a test. Such reference nucleic acid is ideally as similar as possible to the test sample, is available containing combinations of all relevant mutations and polymorphisms, and also has broad applicability to all test formats. Additionally, the reference nucleic acid should be easily produced, quantitated, and packaged with minimal technical capability. Materials meeting these requirements, however, are not available. Reference materials in use include cultured cell lines and patient-based controls materials such as previously tested DNA. They also include DNA extracted and purified from cell lines or patient based specimens. These materials suffer, however, in that they are expensive, difficult to maintain, and limited with respect to the number of genetic diseases, organisms, and combinations of mutations and polymorphisms that they represent.
The need to rely on patient-derived control material also makes it difficult to provide sufficient reference products to cover the large variety of genetic disorders. This is especially problematic when testing for diseases caused by multiple mutations. For example, cystic fibrosis (CF) is a common hereditary disease affecting 1 in 3200 Caucasian newborns in the United States, but the wide variety of mutant alleles makes it difficult to assemble a comprehensive CF proficiency panel. At the NIST Nucleic Acid Workshop, Wayne Grody, Division of Medical Genetics at UCLA, acknowledged the lack of DNA standards and noted that the CAP/ACMG Biochemical and Molecular Genetics Resource Committee would like to dramatically increase the challenges offered.
Further, the unavailability of widely applicable controls is due in part to the variety of different technologies and techniques currently employed for a given diagnostic determination. For example, genetic determinations currently include the use of the polymerase chain reaction (PCR), the ligase chain reaction (LCR), branched DNA, allele specific hybridization, and direct sequence determination. In addition, so-called “home brew” produced primer oligonucleotides, and isotopically labeled or non-radioisotopic based probes are used in a variety of configurations in genetic testing, but without any systematic quality control materials, and hence without any validation.
The aforementioned factors, coupled with the lability of nucleic acids, make it virtually impossible to obtain standard reagents to qualitatively and/or quantitatively assess the overall accuracy, reliability, and efficiency of a laboratory assay.
Cystic fibrosis (CF) is an important genetic disease related to mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Many of the most common disease causing mutations are in exon 10 and exon 11 of the CFTR gene, and thus, genetic screening for these mutations is advantageous for early diagnosis of CF. Genetic testing for CF, as well as many other diseases, typically begins with amplification of the nucleic acid segment of interest (e.g., exon 10 and 11), and therefore, controls for these tests must include the nucleic acid region to be amplified. Quality controls for genetic tests are required by
Gordon Joan
Rundell Clark A.
Horlick Kenneth R.
Maine Molecular Quality Controls, Inc.
Strzelecka Teresa
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