Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-06
2002-04-23
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S063000, C436S002000
Reexamination Certificate
active
06376177
ABSTRACT:
TABLE OF CONTENTS . . .
TABLE OF CONTENTS . . .
1. FIELD OF THE INVENTION . . .
1.1 BRIEF DESCRIPTION OF THE INVENTION . . .
1.2 BACKGROUND OF THE INVENTION . . .
2. SUMMARY OF THE INVENTION . . .
3. BRIEF DESCRIPTION OF THE DRAWINGS . . .
4. DETAILED DESCRIPTION OF THE INVENTION . . .
5. EXAMPLES . . .
6. REFERENCES . . .
THE CLAIMS . . .
ABSTRACT . . .
1. FIELD OF THE INVENTION
1.1 Brief Description of the Invention
The invention disclosed herein relates to a new gene probe biosensor employing near field surface enhanced Raman scattering (NFSERS) for direct spectroscopic detection of DNA hybridization without the need for labels, and the invention also relates to methods for using the biosensor.
1.2 Background of the Invention
In 1928, C. V. Raman and his collaborator, K. S. Krishnan, established that the spectrum of inelastically scattered light can provide a unique fingerprint of molecular structure. Since this initial discovery, Raman spectroscopy has advanced dramatically. Many Raman-related analytical instruments have been developed, some of which have applicability to proteins and nucleic acids. Recent developments have enabled the use of Raman spectroscopy to obtain information such as conformation and/or orientation of molecules and some molecular groups, local hydrogen bonding interactions, and time dependence of structural or organizational properties. Thomas, G. J., “Raman Spectroscopy of Protein and Nucleic Acid Assemblies,”
Annu. Rev. Biophys. Biomol. Struct
. 28:1-27 (1999).
The discrete vibrational energies (Raman band frequencies), scattering probabilities (Raman intensities) and tensor characteristics (Raman polarizations) that constitute the Raman spectra are a function of molecular geometry and intra- and intermolecular force fields.
Early experimental work in the field of Raman spectroscopy demonstrated the advantages of surface-enhanced Raman scattering (SERS) as a technique for detecting and identifying molecules. See Cotton, T. M. “Application of Surface-Enhanced Raman Spectroscopy to Biological Systems”
J. Raman Spect
. 23: 729-742 (1991). For example, between 1974 and 1977, several researchers showed that Raman scattering from pyridine on a roughened silver electrode was enhanced by approximately six orders of magnitude. Id. SERS has been used to study various types of amino acids and peptides on silver surfaces, as well as to study the behavior of DNA at silver colloids.
Surface enhanced Raman scattering has also been investigated as a method for detecting and identifying single base differences in double stranded DNA fragments. Chumanov, G. “Surface Enhanced Raman Scattering for Discovering and Scoring Single Based Differences in DNA” Proc. Volume
SPIE
, 3608 (1999).
SERS has also been used for single molecule detection. Kneipp, K. “Single Molecule Detection Using Surface-Enhanced Raman Scattering (SERS)” Physical Review Letters 78(9):1667-1670 (1997). SERS results in strongly increased Raman signals from molecules which have been attached to nanometer sized metallic structures.
SERS principles have also been used in the development of gene probes which do not require the use of radioactive labels. These probes can be used to detect DNA via hybridization to a DNA sequence complementary to the probe. Vo-Dinh, T. “Surface-Enhanced Raman Gene Probes”
Anal. Chem
. 66:3379-3383 (1994).
The Human Genome Project and other recent advances in molecular biology have spurred the development of new methods for the labeling and detection of DNA and DNA fragments.
Traditionally, radioisotopes have been used as labels for DNA. More recently, fluorescent, chemiluminescent and bioactive reporter groups have been used. The reporter groups are typically incorporated in the primers or the deoxynucleoside triphosphates to label the newly synthesized DNA fragments. The DNA fragments of interest are allowed to hybridize to a set of bound or immobilized DNA fragments.
Among the various methods for identifying genes, the most widely used are technologies which require radioactive labels. A variety of disadvantages are associated with the use of radioactive labels, including the short shelf life of common labels and the safety hazards associated with the use of radioactive compounds. Accordingly, there is a strong need in the art for a method for identifying genes which does not require the use of radioactive labels.
Methods for manufacturing oligonucleotide, DNA and protein microchips and microarrays are known in the art. Research is ongoing into the use of such microchips and microarrays in DNA and RNA sequence analysis, diagnostics of genetic disease, gene polymorphisms studies, and analysis of gene expression. Microchips have been developed in which oligonucleotides are immobilized within polyacrylamide gel pads. Robotics can be employed for the manufacture of microchips containing thousands of immobilized compounds.
Various attempts have been made to enable the sequencing of DNA without the necessity of using radioisotopes, or fluorescent substances. For example, U.S. Pat. No. 5,821,060 describes a process for DNA sequencing, mapping and diagnostics which utilizes the differences between the chemical composition of DNA and that of peptide nucleic acid sequences (PNAs) to provide DNA sequencing, mapping or diagnostics using natural DNA fragments. The process includes the steps of hybridizing PNA segments to complementary DNA segments which are affixed to a hybridization surface, or hybridizing in DNA segments to complementary PNA segments which are fixed to a hybridization surface and using mass spectrometric or non-mass spectrometric techniques to analyze the extent of hybridization at each potential hybridization site.
It is a an object of the present invention to provide molecular sequencing, mapping, screening, diagnostic process and other molecular hybridization processes, in which normal, unlabled DNA is used rather than DNA labeled with stable isotopes, radioactive isotopes or fluorescent groups, and which provides superior spectral specificity as compared to methods of the prior art. Achieving this object will eliminate some of the expensive reagents and labor involved in the labeling of DNA and thereby significantly reduce time, effort and expense of DNA analysis, while enabling highly accurate DNA sequencing, mapping, screening, diagnostic and other molecular hybridization related processes.
In some cases, polymorphisms comprise mutations that are the determinantive characteristic in a genetic disease (hemophilia, sickle-cell anemia, etc.). A “polymorphism” is a variation in the DNA sequence of some members of a species. A polymorphism is said to be “allelic” because some members of a species have the mutated sequence, while other members have the non-mutated sequence. Single nucleotide polymorphisms (SNPs) contain a polymorphic site. A variety of methods have been developed for the characterization of SNPs. Such methods include, for example, the direct or indirect sequencing of the site, the use of restriction enzymes with specificity for the allelic site to create or destroy a restriction site, the use of allele-specific hybridization probes, the use of antibodies that are specific for the proteins encoded by the different alleles of the polymorphism, and other biochemical techniques. It is an object of the present invention to provide advanced surface detection methods which enable the characterization of SNPs without the necessity for the use of restriction enzymes which affect the SNP site, without the necessity for allele-specific hybridization probes, and without the necessity of using antibodies specific for the proteins encoded by the different alleles of the polymorphism.
Other objects and advantages of the present invention over the prior art will become apparent to those skilled in the art upon review of the detailed description that follows.
2. SUMMARY OF THE INVENTION
The applicant has surprisingly and unexpectedly discovered, using a novel analytic technique, coupling near-field optics with SERS techniques, that each hybridization member in
Barrett William A.
Brusca John S.
Hultquist Steven J.
Lundgren Jeffrey S.
Virtual Pro Inc.
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