Nucleic acid amplification and detection method

a nucleic acid and detection method technology, applied in the field of nucleic acid chemistry, can solve the problems of limiting the sensitivity of this latter, affecting the cost-effectiveness of industrial-scale production, and affecting the development of microarrays

Inactive Publication Date: 2005-12-29
GEN PROBE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018] As used herein, “microparticles” are solid or semi-solid particles having a diameter of less than one millimeter, more preferably less than 100 microns, still more preferably less than 10 microns, which can be formed of a variety of materials, including glass or plastic, as well as derivatized glass or plastic. Preferred microparticles are monodisperse (i.e., uniform in size within about 5%), thereby providing consistent results which are particularly advantageous for use in an automated assay.

Problems solved by technology

Unfortunately, a stoichiometric relationship between the number of arrayed primers immobilized on the chip and the number of extension products available for detection limits the sensitivity of this latter, microarray-based technique.
While protein microarrays represent an emerging technology, their development has been hampered somewhat because the active conformations of complex proteins have been difficult to preserve through the necessary immobilization procedures.
One of the challenges underlying efficient production of microarrays relates to the immobilization chemistry that joins the deposited macromolecules to the solid substrate.
While convenient, this approach may not be cost-effective for industrial-scale production.
Other approaches based on covalent immobilization chemistries are frequently burdened by complicated synthetic routines that lead to inefficiencies in the yield of desired products.

Method used

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Examples

Experimental program
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Effect test

example 1

Efficient Introduction of Nucleophiles onto Glass Surfaces

[0073] Glass-bottomed polystyrene 96-well plates (Whatman Polyfiltronics; Rockland, Mass.) were cleaned by soaking in a solution of 10% H2O / 50% MeOH in water for 30 minutes at room temperature, washed with water three times using an automated plate washer, and then dried in a vacuum desiccator for at least 2 hours. Cleaned and dried plates were contacted with 100 μl per well of different concentrations of 3-(mercaptopropyl)trimethoxysilane (3 MPS) in various solvent mixtures for different times at room temperature (about 23° C.), washed manually three times with 100 μl of 90% MeOH, washed three times with 100 μl water using the automated plate washer, and then vacuum desiccated overnight. Wells (n=12) from the 120 minute time point were used subsequently for studying nonspecific binding. Coupling reactions were carried out by contacting the silanized surfaces for 60 minutes at room temperature with 25 pmol of a synthetic oli...

example 2

Coupling Oligonucleotides to Glass or Plastic Microparticles

[0077] Batches of magnetic porous glass (MPG) 5 μm microparticles obtained from CPG, Inc., (Lincoln Park, N.J.) were allowed to react with 3 MPS or γ-aminopropyltrimethoxysilane (GAPS) and subsequently coupled with a fluorescent oligonucleotide. In this procedure GAPS was used for introducing functional amine groups instead of sulfhydryl groups onto different surfaces. Uncoated 5 μm Magnetic Porous Glass microparticles had nominal surface areas of 314 μm2 per particle. Sulfhydryl derivatized 1.46 μm ESTAPOR polystyrene magnetic microparticles from Merck Eurolab S.A., (France) had nominal surface areas of 6.70 μm2 per particle. Using either 3.60×106 glass or 1.69×108 polystyrene microparticles per ml, samples were washed 3 times using 1 ml volumes of 90% MeOH, and placed in vacuum desiccator to dry. Separate aliquots of glass microparticles were suspended in 4% 3 MPS or GAPS dissolved in 90% MeOH, incubated for 2 hours with...

example 3

Effect of pH Range and Buffer Type on Oligonucleotide Coupling

[0080] A glass-bottomed polystyrene 96 well plate was cleaned and then dried overnight as described under Example 1. Wells of the cleaned plate were contacted with 100 μl per well of 4% 3 MPS or 4% GAPS dissolved in 90% MeOH at room temperature for 120 minutes, then washed manually three times with 90% MeOH, washed with an automated plate washer three times with water, and vacuum desiccated overnight. Wells from the 120 minute timepoint (n=12) were subsequently used for studying nonspecific binding of oligonucleotides. Coupling reactions were performed using 25 pmol of the carboxylated and fluoroscein-labeled oligonucleotide from Example 1 in 100 μl volumes of a solution made 0.1 M of the indicated buffer, 1% PEG, and 0.1 M EDAC for 60 minutes at room temperature. EDAC was omitted from control trials that monitored nonspecific reactions. Wells were soaked in 1× TENT buffer (for 3 MPS-treated surfaces) or in a solution of...

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Abstract

Method of amplifying and detecting a nucleic acid target molecule contained in a test sample using a device having amplification primers and detectably labeled hybridization probes immobilized on the same surface of a solid support. Soluble amplicons produced in an in vitro nucleic acid amplification reaction employing the immobilized amplification primers are detected by the immobilized hybridization probes. Surface-immobilized amplification primers and hybridization probes are maintained in fluid communication throughout the procedure.

Description

RELATED APPLICATIONS [0001] This application is a continuation of application Ser. No. 10 / 621,803, filed Jul. 17, 2003, which claims the benefit of U.S. Provisional Application No. 60 / 400,189, filed Jul. 31, 2002. The entire disclosures of these related applications are hereby incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates to the field of nucleic acid chemistry. More specifically, the invention relates to methods of making and using devices that incorporate immobilized oligonucleotides. BACKGROUND OF THE INVENTION [0003] The molecular diagnostics industry has been revolutionized by the advent of nucleic acid amplification technology. Enzyme-based nucleic acid amplification is conventionally practiced using at least two oligonucleotide primers, each primer being complementary to an opposite strand of the nucleic acid that is to be amplified. Also conventionally, the primers are soluble and able to diffuse freely through solution to encounter a c...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): B01J19/00C40B60/14
CPCB01J19/0046C40B60/14B01J2219/005B01J2219/00529B01J2219/00576B01J2219/00608B01J2219/0061B01J2219/00612B01J2219/00621B01J2219/00626B01J2219/0063B01J2219/00637B01J2219/00677B82Y30/00C07B2200/11C40B30/04C40B40/06C40B50/14B01J2219/00313
Inventor BROWNE, KENNETH
Owner GEN PROBE INC
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