Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-08
2002-08-27
Whisenant, Ethan C. (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024330, C536S025300, C536S025320, C435S006120, C435S091100
Reexamination Certificate
active
06441152
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is related to the field of probe-based detection, analysis and quantitation of nucleic acids which are electrostatically immobilized to matrices. The methods, kits and compositions of this invention are particularly well suited for the analysis, and particularly single point mutation analysis, in a particle assay, in an array assay, in a nuclease digestion/protection assay, in a line assay and/or in a self-indicating assay format.
2. Description of the Related Art
Nucleic acid hybridization is a fundamental process in molecular biology. Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids. Nucleic acid probes have long been used to analyze samples for the presence of nucleic acid from bacteria, eucarya, fungi, virus or other organisms and are also useful in examining genetically-based disease states or clinical conditions of interest in single cells as well as in tissues.
Sample prep methods which describe the repetitive capture and release of target sequences to and from supports (e.g. magnetic beads) as a means to remove non-target polynucleotides, debris and impurities which tend to introduce background in a hybridization assay are known in the art (See: Collins et al., U.S. Pat. No. 5,750,338). Generally, the sample prep methods of Collins et al. can be used in most embodiments of traditional hybridization assays provided however that the target nucleic acid is first immobilized to a support and thereafter released from the support such that, when released, it is substantially free of sample impurities, debris, and extraneous polynucleotides. The Collins et al. invention, however, requires that the probe or probes must be associated with or capable of associating with the support under binding conditions to thereby immobilize the nucleic acid of interest to the support (See: Collins et al. at col. 4, line 55 to col. 5, line 13).
A probe based sample prep method for removing contaminants prior to PCR reaction has been described by Goldin et al. (See: U.S. Pat. No. 5,200,314). This process requires an analyte-capture probe having both an analyte binding region and a first specific binding partner. Like the Collins et al. invention, the Goldin et al. invention requires that the analyte-capture probe interact with the support as the specific means through which the target sequence becomes immobilized.
Polycationic solid supports have been used for the analysis and purification of nucleic acids, including the purification of polynucleotides from solutions containing contaminants See: Arnold et al.; U.S. Pat. No. 5,599,667). Arnold et al. describe assays which use solid supports as a means to separate polynucleotides, and hybrids thereof formed with a nucleotide probe, from unhybridized probe (See: Abstract to U.S. Pat. No. 5,599,667). The invention is premised upon “. . . the discovery that polycationic solid supports can be used to selectively adsorb nucleotide multimers according to their size (emphasis added), larger multimers being more tightly bound to the support than smaller ones.” (See: Col. 4, lines 39-44). The methods can also be used to separate the nucleotide multimers from non-nucleotidic material (See: Col. 5, lines 25-28).
A substantial limitation of the Arnold et al. invention is the interplay which exists between the composition of the cationic solid support and the formulation of contacting solutions as well as the interplay between two or more of the contacting solutions (See: Col. 7, line 24 to Col. 8, line 32) which are required to discriminate between nucleotide multimers (See: Col. 8, lines 39-41). An example of a laborious protocol for arriving at a proper cation density for a solid support can be found at col. 9, lines 36-52 and the method for determining the buffer concentration suitable for separating polynucleotides and nucleotide probes can be found at col. 9, lines 53-63. Similarly, the separation solution must be carefully designed (See: Col. 10. lines 9-12), presumably using the laborious method of trial and error as described for determining the cation density of the solid support. This requirement for substantial optimization of assay conditions within a very narrow operating range results because electrostatic immobilization of nucleic acid is a relatively non-specific process and therefore it is difficult to electrostatically immobilize a negatively charged target nucleic acid to a cationic surface without the positively charged matrix also exhibiting a strong affinity for the negatively charged nucleic acid probe. Since the separation of nucleotide multimers (nucleotide probe/target hybrids from excess nucleotide probe) occurs within a narrow range of conditions, which may not necessarily be optimal for the discrimination of hybridization, the hybrids still immobilized according to the Arnold et al. invention may not be truly indicative of the presence of a target sequence. Consequently, the applicability of the assays of Arnold et al. are of limited practical utility.
An invention related to achieving nucleic acid has recently been described (See: Gerdes et al.; WO98/46797). Gerdes et al. use highly electropositive solid phase materials to capture nucleic acids (See. p. 5, line 24 to p. 6, line 14) for repetitive analyses. However, a substantial limitation of the Gerdes et al. invention is that the nucleic acid must be irreversibly bound to the highly electropositive solid phase material.
Methods for the high throughput screening for sequences or genetic alterations in nucleic acid have been described (See: Shuber, A. P.; U.S. Pat. No. 5,834,181). Shuber describes the analysis of arrays of immobilized nucleic acids, and suggests immobilization of the nucleic acid to nitocellulose or a charged nylon membrane (See: col. 6, lines 41-64). Suggested purine and pyrimidine containing polymers which may be used for analyzing immobilized nucleic acid include peptide nucleic acid (See: col. 5, lines 15-20), but the polymers must necessarily be tagged or labeled since the detection methods rely on a tag or label being incorporated into the polymer (See: col. 8, line 58 to col. 9, line 3). The assays of Shuber require a perfect complement between probe and target sequence (See: col. 8, lines 52-57). In order to achieve proper discrimination, a laborious empirical process of trial and error is described for assay optimization (See: col. 7, line 16 to col. 8). Conditions which require optimization of specific and non-specific hybridization include the concentration of polymer, the temperature of hybridization, the salt concentration, and the presence or absence of unrelated nucleic acid (See: col. 8, lines 15-18).
Shuber does not expressly suggest performing a probe-based hybridization assay on an electrostatically immobilized nucleic acid and specifically does not describe or teach the analysis of electrostatically immobilized nucleic acid using a non-nucleotide probe such as a peptide nucleic acid. Furthermore, Shuber does not suggest, disclose or teach any advantages, such the ability to work within a broad range of assay conditions, of performing a peptide nucleic acid-based analysis of nucleic acid electrostatically immobilized to a matrix.
Pluskal et al. describe a comparison of DNA and peptide nucleic acid (PNA) probe-based analysis of nucleic acid which has been irreversibly crosslinked to charged nylon membrane (See: Pluskal et al., American Society for Biochemistry, 85th Annual Meeting, Washington, D.C., May 1994). Pluskal et al. teach that while PNA probes can be used to detect the irreversibly immobilized nucleic acid under standard hybridization conditions, PNA works very well under highly stringent hybridization and washing conditions (See: The Section Entitled “Discussion”). Pluskal et al. also teach the use of 1% BSA as a blocking agent to reduce non-specific binding of the probe to the membrane (See: Section Entitled “Discussion”). Because the nucleic acid of Pluskal et al. has been irreversibly crosslinked to the nylon membrane, h
Coull James M.
Fiandaca Mark J.
Hyldig-Nielsen Jens J.
Johansen Jack T.
Boston Probes, Inc.
Gildea Brian D.
Lu Frank
Whisenant Ethan C.
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