Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-21
2002-03-12
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S287200, C435S288700, C436S164000, C436S172000, C536S063000, C536S063000, C536S024300, C536S023100
Reexamination Certificate
active
06355429
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to fields using devices and methods for detecting nucleic acid hybridization.
BACKGROUND OF THE INVENTION
The following is a discussion of relevant art, none of which is admitted to be prior art to the appended claims.
Nucleic acid probe technology has application in detection of infectious disease and genetic and cancer screening. Nucleic acid based probe methods offer several advantages over conventional microbiological or immunological methods for detection of organisms, as described by Nakamura and Bylund (
J. Clinical Laboratory Analysis
, 6, 73-83, 1992).
Methods to amplify either the number of copies of the nucleic acid available for detection or the signal generated after hybridization of the nucleic acid probe have been utilized. A review of nucleic acid based detection methods and various amplification schemes such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription based amplification, cycling probe reaction, Q&bgr; replicase, and strand displacement amplification may be found in M. J. Wolcott,
Clinical Microbiology Reviews
, 5, October 1992, pp 370-386.
U.S. Pat. No. 5,175,270 describes an amplification reagent consisting of layers of nucleotide polymers containing double stranded and single stranded sections. Each section has an end which is capable of hybridizing with another molecule.
Probe or hybridization assays are often based on the attachment of an oligonucleotide probe to a surface in order to capture a target nucleic acid molecule (analyte) from a sample. The attachment of this probe to the surface may be through covalent bonds or through a variety of passive absorption mechanisms (e.g., hydrophobic or ionic interactions).
U.S. Pat. No. 5,279,955 describes an immobilization process which uses a heterofunctional cross-linker for a plastic support. The cross-linker consists of a central ring which is hydrophobic and interacts with the plastic, and a hydrophilic chain which terminates in a group capable of reacting with a nucleic acid. Covalent attachment is achieved through a succinyl-olivetol-N-hydroxysuccinimide.
U.S. Pat. No. 5,262,297 describes immobilization of a probe through co-polymers which contain reactive carboxylic acid groups and an 8-50 atom spacer with two or more unsaturated groups within the spacer.
U.S. Pat. No. 5,034,428 describes an immobilization process for probes in which a monomer is joined onto a hydrophilic solid support which can be irradiated in an oxygen free atmosphere. This method provides for covalent attachment of the probe.
U.S. Pat. No. 4,806,546 also describes an immobilization process for an amide modified nylon. The method relies on an amidine linkage under anhydrous conditions in the presence of an alkylating agent.
Maskos and Southern, 20 Nucleic Acids Research 1679, 1992, describe a linker system for the attachment of a nucleic acid to a glass support. The linker system allows for the chemical synthesis of a strand of nucleic acids on the surface. The primary advantage of the linker is that it is stable to an ammonia treatment which is required in the synthesis of the polynucleotide. A hexaethylene glycol spacer is incorporated into the linker which attaches to the glass through a glycidoxypropyl silane which terminates in a primary hydroxy group. The silane is condensed onto silane groups on a solid support. Additional cross-linking may be obtained by introducing water so that the epoxide group is cleaved to a diol. An acidic solution facilitates this process. The length of the linker may be varied by changing the spacer to ethylene glycol, pentaethylene glycol, etc.
Nucleic acid probes that have hybridized to their target sequence are detected based on various methods that introduce a detectable chemiluminscent, fluorescent or other identifiable label into a nucleic acid probe. Several of these techniques are described in U.S. Pat. Nos. 4,968,602, 4,818,680, 5,104,791, and 5,272,056, and International applications WO91/00926 and GB2169403A.
Arnold et al., U.S. Pat. No. 5,283,174 describe the use of a chemiluminscent label with DNA probes. The label is composed of an acridinium ester and has a number of desirable properties. It is stable to hybridization conditions, light is emitted only upon exposure to an alkaline peroxide, the emission kinetics are rapid, and the label on the unhybridized probe can be destroyed without an impact on the signal generated by hybridized probe.
U.S. Pat. No. 5,089,387 describes a diffraction assay for the detection of DNA hybridization. In this invention, a solid support, generally silicon or polysilicon, is coated with a DNA probe. These surfaces are required to inherently adhere the DNA probe to the surface. Once the surface is coated with the probe, the surface is selectively inactivated to provide a series of very strictly controlled reactive probe lines for the generation of the diffraction grating. The unreacted surface is required to be non-light disturbing. The diffraction grating is only generated upon the addition of the analyte to the surface. The angle of diffraction is a function of the wavelength of incident light and the density and spacing of the individual gratings on the surface. A single detector or a multiple detector array may be used to detect and measure the light from all desired orders of the diffracted light.
Mixed phase systems have typically been used to perform hybridization assays. In mixed phase assays the hybridizations are performed on a solid phase such as nylon or nitrocellulose membranes. For example, the assays usually involve loading a membrane with a sample, denaturing the DNA or creating single stranded molecules, fixing the DNA or RNA to the membrane, and saturating the remaining membrane attachment sites with heterologous nucleic acids to prevent the probe reagent from adhering to the membrane in a non-specific manner. All of these steps must be carried out before performing the actual hybridization.
SUMMARY OF THE INVENTION
This invention features improved devices, and methods for producing and using optical devices, for detecting the presence or amount of a specific target nucleic acid within a sample. The current invention is based on a probe coated substrate which is optically active. Surfaces can be pre-selected for the type of optical thin film detection to be employed, and enable direct detection of the hybridization reaction through the interaction of light with thin films. Detection of specific target nucleic acid sequences is also referred to as sequence analysis.
This invention also describes materials and methods for producing optically active solid supports or devices for use in nucleic acid hybridization assays and immobilization of nucleic acid probes to such surfaces. These surfaces are compatible with a wide range of optical thin film detection methods, all of which utilize some interaction of thin films with light. Such optical thin film detection methods include optical interference, ellipsometry—comparison, null, photometric and other modifications, attenuation of polarized light at non-Brewster angles, profilometry, scanning tunneling microscopy, surface plasmon resonance, evanescent wave techniques, reflectometry, or atomic force microscopy.
The direct optical thin film detection methods of the current invention are extremely sensitive to changes in mass at the surface of an optically active substrate. These optical thin film detection methods provide increased sensitivity for hybridization assays without the introduction of signal generating labels or pre-assay target amplification with its accompanying complexity and limitations. Thus, assay protocols utilizing optical thin film detection methods are greatly simplified, are more rapid, and less costly than conventional indirect assay methods. Total assay times may vary from one hour to a few minutes from the initiation of the assay protocol (i.e., from the time that the target nucleic acid containing sample is contacted with the device). Such devices also allow for assay results to be visu
Nygren Hakan
Stenberg Manne
Foley & Lardner
Myers Carla J.
Thermo Biostar Inc.
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