Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1993-05-13
1995-12-12
Fleisher, Mindy B.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 243, 536 2431, 536 2532, 435 912, 435 9121, C12Q 168, C12N 1511
Patent
active
054748950
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to means for the detection of nucleic acid sequences. In particular, the invention relates to bead-based hybridization assay systems that are useful for the capture and detection of nucleic acids, including RNA and single-stranded DNA targets. In another aspect, this invention relates to kits and compositions for practicing the bead-based hybridization assays.
BACKGROUND OF THE INVENTION
It is often desirable to detect very small amounts of nucleic acids, such as samples obtained from biological samples. According to one common approach, nucleic acids, target nucleic acids, are extracted from the sample and are hybridized to an oligonucleotide to form a detectable complex. In order to obtain a detectable signal that can be correlated with the amount of the target, either the target nucleic acid or the oligonucleotide is associated with a signal generating reporter element, such as a radioactive atom, a fluorescent or chromogenic molecule, or an enzyme which is capable of converting a substrate into a product which can be detected and measured. The signal generated, directly or indirectly, by a properly hybridized nucleic acid is detected and measured by methods known in the art.
Many of the commonly used techniques of molecular biology, including fractionation and identification of specific sequences of nucleotide bases, involve the immobilization of target nucleic acid sequences on solid supports. For example, target nucleic acid sequences have been immobilized on nylon or nitrocellulose membranes, then detected with an oligonucleotide having a radioactive label attached thereto. Alternatively, so-called "sandwich" hybridization systems may be employed, using a capture oligonucleotide, which includes a sequence of nucleotide bases homologous to or complementary to the target and which is covalently attached to, or non-covalently associated with, a solid support, and using a detection oligonucleotide, which is an oligonucleotide covalently attached to, or non-covalently associated with, a reporter group, such as a radioactive label or a detection enzyme, and that has a sufficient complementarity with the target nucleic acid sequence in a region that is different from that portion of the target nucleic acid sequence which hybridizes to the capture oligonucleotide under conditions such that a dual hybridization occurs with the target sequence. The capture oligonucleotide, target nucleic acid and detection oligonucleotide form a sandwich complex by hybridization of the target with both the capture and detection oligonucleotide.
The solid support may be in the form of beads in which case the assay is referred to as a "bead-based sandwich hybridization system" (herein referred to as a BBSHS). A BBSHS is described, for example, in European Patent Application No. 276,302. According to this method, in a first step, the target nucleic acid and an oligonucleotide probe used for its detection, which is complementary to a first region of the target, are hybridized. The complex thus formed is then captured by a second oligonucleotide that is complementary to a different region of the target. In addition, the capture oligonucleotide is preferably end-attached to a solid support. The amount of the detection oligonucleotide associated with the solid support after these hybridization steps is directly related to the amount of the target nucleic acid captured. In this way, the BBSHS can be used to determine the amount of a specific single-stranded nucleic acid in a sample. In this and similar assays, radioactively (e.g., .sup.32 P) labeled cloned DNAs or synthetic oligonucleotides are most commonly employed because of the high sensitivity which can be obtained with such labels. .sup.32 P-labeled oligonucleotide probes used in conjunction with SEPHACRYL.TM. or TRISACRYL.TM. (Sepracor Inc.) beads in BBSHS experiments provide about 10:1 or better signal to noise ratios with target sequences present in about 0.5 mole amounts.
In practice, because of the inconveniences ass
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Ghosh Soumitra
Ishii Jennifer K.
Carter Philip W.
Fleisher Mindy B.
Seidman Stephanie L.
Siska Diagnostics Inc.
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