Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1992-03-03
1995-09-19
Parr, Margaret
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
439294, 439290, 439289, 935 87, C12Q 170
Patent
active
054515009
DESCRIPTION:
BRIEF SUMMARY
This application is the national filing of PCT application No. PCT/US90/06768 filed Nov. 16,1990, claiming priority of U.S. patent application Ser. No. 07/438,592 filed Nov. 17, 1989, now U.S. Pat. No. 5,188,963.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method and apparatus, including component devices, for use in automating the detection of target nucleic acid sequences in biological-containing samples. The method involves a sequence of physical and chemical reactions, and more particularly to a system for the exposure of, amplification of, and labelled-probe coupling to, a specific, known nucleic acid sequence. The process of the invention consists of the following stages: 1) matrix dispensing, sample mixing and DNA immobilization; 2) preparing DNA; 3) amplifying DNA target sequences; 4) hybridizing a labeled probe to the target; and 5) scanning the matrices for signal produced by bound label.
The invention is especially suited to the automated detection of single, specific genetic sequences present at random in multiple samples containing biological material without labor-intensive DNA extraction and purification procedures being performed separately on each sample. The ability to detect single copies of a specific nucleic acid in biological or environmental samples makes this process revolutionary.
2. Description of the Related Art
There is no laboratory apparatus or equipment currently on the market that automates DNA preparation, modification and detection in one, unattended operation. The apparatus and devices described herein embody an automated process, including a fluid-delivery system and a thermal reaction chamber.
Devices for receiving biological specimens for diagnostic purposes are varied and adapted to the methods of detection. The devices may take the form of tubes for liquid specimens, flat surfaces such as glass slides suitable for microscopy, microtiter dishes, Petri dishes and cubes containing growth medium, or filters made of various materials to which cell and viral components will adhere.
These specimen samples are then treated in such a way as to indicate either the presence or absence, or quantity, of a specific biological entity. Test reagents may either be preapplied to the device or added in series after the specimen is present. Test results are read manually by a technical person or automatically with instrumentation specifically designed for that assay. In some instances the specimen is diluted with a diluent, or an aliquot of the specimen is removed from the original collecting device and transferred to another vessel at some point in the assay. In some cases physical and chemical means are used to amplify the signal of the assay for greater sensitivity. Some assays require extraction or separation to isolate a specific component from other parts.
In DNA-based diagnostics the sequence specificity of base-pairing or enzymatic or other types of cleavage is exploited. The linear sequence of nucleotides in double-stranded DNA molecules forms the basis of replication of the genetic code. Hybridization is the binding of two single-stranded DNA strands whose base-pairing sequences are complementary. Temperature and salt concentration affect the stringency of these base-pairing matches. A change from high stringency to low stringency can cause the same DNA probe to be either exquisitely specific to detect a particular target or less specific and detect a group of related targets.
In some instances the sizes of DNA fragments, produced by restriction endonuclease digestion or by amplification of a target sequences between primer pairs, are used to make a DNA-print for individual identification or aid in diagnosis of a genetic disease, cancer or infectious disease. For example, electrophoresis may be used to size-fractionate different-sized nucleic acids which have been specifically cleaved or whose native length puts them in a distinguishable size-length class.
In the electrophoresis method, a current is applied to DNA loaded at the cathodal
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Gene Tec Corporation
Houtteman Scott W.
Parr Margaret
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