Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-29
2002-10-22
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C536S023100, C536S024330, C250S281000, C250S282000, C250S283000, C250S288000, C250S287000, C210S635000, C210S656000
Reexamination Certificate
active
06468748
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to methods for screening nucleic acids for mutations by analyzing fragmented nucleic acids using mass spectrometry.
INTRODUCTION
Approximately 4,000 human disorders are attributed to genetic causes. Hundreds of genes responsible for various disorders have been mapped, and sequence information is being accumulated rapidly. A principal goal of the Human Genome Project is to find all genes associated with each disorder. The definitive diagnostic test for any specific genetic disease (or predisposition to disease) will be the identification of mutations in affected cells that result in alterations of gene function. Furthermore, response to specific medications may depend on the presence of mutations. Developing DNA (or RNA) screening as a practical tool for medical diagnostics requires a method that is inexpensive, accurate, expeditious, and robust.
Genetic mutations can manifest themselves in several forms, such as point mutations where a single base is changed to one of the three other bases, deletions where one or more bases are removed from a nucleic acid sequence and the bases flanking the deleted sequence are directly linked to each other, and insertions where new bases are inserted at a particular point in a nucleic acid sequence adding additional length to the overall sequence. Large insertions and deletions, often the result of chromosomal recombination and rearrangement events, can lead to partial or complete loss of a gene. Of these forms of mutation, in general the most difficult type of mutation to screen for and detect is the point mutation because it represents the smallest degree of molecular change. The term mutation encompasses all the above-listed types of differences from wild type nucleic acid sequence. Wild type is a standard or reference nucleotide sequence to which variations are compared. As defined, any variation from wild type is considered a mutation including naturally occurring sequence polymorphisms.
Although a number of genetic defects can be linked to a specific single point mutation within a gene, e.g. sickle cell anemia, many are caused by a wide spectrum of different mutations throughout the gene. A typical gene that might be screened using the methods described here could be anywhere from 1,000 to 100,000 bases in length, though smaller and larger genes do exist. Of that amount of DNA, only a fraction of the base pairs actually encode the protein. These discontinuous protein coding regions are called exons and the remainder of the gene is referred to as introns. Of these two types of regions, exons often contain the most important sequences to be screened. Several complex procedures have been developed for scanning genes in order to detect mutations, which are applicable to both exons and introns.
Gel Electrophoresis
Several of the procedures described below use some form of gel electrophoresis. Therefore it is worthwhile to briefly consider this separation technology before proceeding to the specific methods. In terms of current use, most of the methods to scan or screen genes employ slab or capillary gel electrophoresis for the separation and detection step in the assays. Gel electrophoresis of nucleic acids primarily provides relative size information based on mobility through the gel matrix. If calibration standards are employed, gel electrophoresis can be used to measure absolute and relative molecular weights of large biomolecules with some moderate degree of accuracy; even then typically the accuracy is only 5% to 10%. Also the molecular weight resolution is limited. In cases where two DNA fragments with identical number of base pairs can be separated, using high concentration polyacrylamide gels, it is still not possible to identify which band on a gel corresponds to which DNA fragment without performing secondary labeling experiments. Gel electrophoresis techniques can only determine size and cannot provide any information about changes in base composition or sequence without performing more complex sequencing reactions. Gel-based techniques, for the most part, are dependent on labeling methods to visualize and discriminate between different nucleic acid fragments.
DNA Sequencing
The principal approach currently used to screen for genetic mutations is DNA sequencing. Sequencing reactions can be performed to screen the full genetic target base by base. This process, which can pinpoint the exact location and nature of mutation, requires labeling DNA, use of polyacrylamide gels, and a multiplicity of reactions to assess all bases over the length of a gene, all of which are slow and labor intensive procedures. [J. Bergh et al. “Complete Sequencing of the p53 Gene Provides Prognostic Information in Breast Cancer Patients, Particularly in Relation to Adjuvant Systemic Therapy and Radiotherapy,” Nature Medicine 1, 1029 (1995)].
For DNA sequencing, nucleic acids comprising different exons or small clusters of exons are individually amplified, often using polymerase chain reaction (PCR). The amplifications are normally performed separately although some multiplexing of reactions is possible. The amplified nucleic acids typically range from one hundred to several thousand bases in length. Following amplification, the PCR products can serve as templates for standard dideoxy-based Sanger sequencing reactions. The four different sequencing reactions are run (or for fluorescence detection, one reaction with four different dye terminators) and then analyzed by polyacrylamide gel electrophoresis. Each sequencing run yields about 300 to 600 bases of sequence which typically must be read with at least a two to three-fold redundancy in order to assure accuracy. Using slab gel, the analysis process typically takes several hours.
SSCP
The single strand conformational polymorphism assay takes advantage of structural variation within DNA that results from mutation. The method involves folding the single-stranded form of a given nucleic acid sequence into a thermodynamically directed secondary and tertiary structure. In most cases, mutated sequences form different structures than the wild type sequence, thus permitting separation of mutated and wild type sequences by gel electrophoresis. Like sequencing, this assay is complicated by the need to label molecules and run polyacrylamide gels. In a typical case, mutations can be located within a general range of 50 to 200 base pairs, but the exact nature of the mutation cannot be identified. [M. Orita et al., “Detection of Polymorphisms of Human DNA by Gel Electrophoresis as Single-Stranded Conformation Polymorphisms,” Proc. Natl. Acad. Sci. USA 86, 2766 (1989)].
DGGE
Like SSCP, denaturing gradient gel electrophoresis assays also differentiate based on structural variation, but require the use of gradient gels, which are difficult to prepare. The different thermodynamic stability of structures formed by the mutant sequence, as opposed to wild type, lead to differences in the temperature and/or pH at which the molecule will denature. DGGE mutation identification and localization properties are similar to those for SSCP though sensitivity is higher for DGGE because not all mutations cause the structural changes that the SSCP method depends upon for detection. [E. S. Abrams, S. E. Murdaugh & L. S. Lerman, “Comprehensive Detection of Single Base Changes in Human Genomic DNA Using Denaturing Gradient Gel Electrophoresis and a GC Clamp,” Genomics 7, 463 (1990)].
EMC
Enzyme mismatch cleavage utilizes one or more enzymes that are capable of recognizing interruptions in base pairing within a double-stranded nucleic acid molecule, e.g. base-base mismatches, bulges, or internal loops. A given length of DNA or RNA is prepared in heterozygous form, with one strand composed of wild type nucleic acid and the other strand containing a potential mutation. At the specific site where the mutation forms a mismatch with the wild type sequence, a structural perturbation occurs. An enzyme such as T4 Tendonuclease VII, RuvC, RNase A, or MutY, can recognize su
Becker Christopher H.
Monforte Joseph A.
Shaler Thomas A.
Tan Yuping
Heller Ehrman White & McAuliffe LLP
Seidman Stephanie
Sequenom Inc.
Siew Jeffrey
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