Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Patent
1990-01-24
1992-12-29
Warden, Robert J.
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
422 71, 422 80, 422116, 436 59, 436155, 436161, 250281, G01N 2306, G01N 3112, G01N 3002
Patent
active
051749629
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates to the field of the determination of DNA sequences and the uses of automated techniques for such determination.
The ability to sequence DNA has become a core technology in molecular biology, and has contributed greatly to the understanding of DNA structural organization and gene function. The facility with which DNA sequencing may be accomplished will substantially affect the rate of development of related technologies, including the production of new therapeutic agents, useful plant varieties and microorganisms via recombinant DNA technology and the understanding of human genetic disorders and pathology through gene mapping and chromosomal sequence analysis.
Initially, researchers focused on reading the genetic code and the translating of the nucleotide sequence into the amino acid sequence of a protein. This occurs by a process of DNA transcription into mRNA, and then actual synthesis of the protein on ribosomes. In eucaryotic cells, large specific segments of the initial transcript of mRNA, termed introns, are transcribed but are excised during an intermediary processing step. Much of the chromosomal DNA is not translated, and its specific function is largely unknown. This "intervening" or intron DNA was first thought to be excess genetic material. However, as biologists begin to unravel the details of cell differentiation and the processes controlling gene transcription it is now believed that the specific sequences of certain portions of some of these large regions of transcribed but untranslated DNA may also provide important regulatory signals.
The potential applications which derive from DNA sequencing have only begun to be explored. On large scale, analysis of human chromosomal DNA is considered vital to understanding human pathological conditions, including genetic disease, AIDS and cancer, because often only subtle differences, even single nucleotide substitutions, can lead to serious disorders. Serious consideration is now being given to the sequencing of the entire human genome--approximately 3 billion base pairs. The success of this project will depend on rapid, sensitive, inexpensive automated methods to sequence DNA.
The fundamental approach to determination of DNA sequence has been well established. Restriction endonucleases are employed to cleave chromosomal DNA into specific smaller segments, and recombinant cloning techniques are then used to purify and generate analyzable quantities of DNA. The specific sequence of each segment can then be determined by either the Maxam-Gilbert chemical cleavage, or preferably, the Sanger dideoxy terminated enzymatic method. In either case, a set of all possible fragments ending in a specific base are generated. The individual fragments can be resolved electrophoretically by molecular weight, and the sequence on the original DNA segment is then derived by knowing the identify of the terminal base in each fragment.
In its broadest aspect, this invention is directed to methods and reagents for sequencing DNA and other polynucleotides. In particular, this invention describes reagents and methods for automating and increasing the sensitivity of both the Sanger, Proc. Natl. Acad. Sci. USA, 74, 5463 (1977) and Gish and Eckstein, Science, 240, 1520-1522 (1988), procedures for sequencing polynucleotides. The methods of the present invention are based on mass spectrometric determination of each of the four component terminal nucleotide residues, where the information regarding the identity of the individual nucleotides is contained in the mass of stable nuclide markers.
2. Summary Of The Prior Art
In the Sanger dideoxy method (Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)), the DNA to be sequenced is exposed to a DNA polymerase, a cDNA primer, and a mixture of the four component deoxynucleotides, plus one of the four possible 2,3-dideoxy nucleotides. The DNA to be sequenced is typically a single stranded DNA clone prepared in the phase vector M13, although Chen and Seeburg have disclosed a method for applying t
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Genomyx Inc.
Trembley T. A.
Warden Robert J.
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