Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-05-05
2002-06-18
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C065S409000, C250S458100, C422S068100, C422S082050, C422S082070, C422S082080, C422S082090
Reexamination Certificate
active
06406845
ABSTRACT:
FIELD OF THE INVENTION
The present application is concerned generally with apparatus and methods for the analysis of genes and gene expression; and is particularly directed to the construction and use of a fiber optic biosensor able to detect selectively one or multiple DNA, RNA, or PNA oligonucleotide fragments concurrently.
BACKGROUND OF THE INVENTION
In less than twenty years, the field of molecular genetics, including the specialty of genetic engineering, has revolutionized the science of biology as a whole and is in the process of restructuring medicine in both diagnostic and therapeutic applications. Not only are individual genes now being isolated and characterized, but also extensive research studies as to how genes function and are regulated in-vivo are being actively pursued. Moreover, many techniques for manipulating and modifying genes have been reported and are today becoming widespread in use and diverse in application. Merely exemplifying the many authoritative texts and published articles presently available in the literature regarding genes, gene manipulation and genetic analysis are the following:
Gene Probes for Bacteria
(Macario and De Macario, editors) Academic Press Inc. 1990
; Genetic Analysis, Principles Score and Obiectives
by John R. S. Fincham, Blackwell Science Ltd., 1994
; Recombinant DNA Methodology II
(Ray Wu, editor), Academic Press, 1995
; Molecular Cloning. A Laboratory Manual
(Maniatis, Fritsch, and Sambrook, editors), Cold Spring Harbor Laboratory, 1982
; PCR
(
Polymerase Chain Reaction
), (Newton and Graham, editors), Bios Scientific Publishers, 1994; and the many references individually cited within each of these publications.
Among the many innovative ideas and novel techniques generated by molecular genetic research studies has been the generation of nucleic acid probes for identifying the existence of specific genes, the products of gene expression, and the presence of mutations in one or more genes. By definition, a nucleic acid probe is a DNA or RNA oligonucleotide fragment or peptide nucleic acid (PNA) of known base sequence. Existing as a single-stranded segment of base codons, a nucleic acid probe which will bind to a complementary base sequence of nucleic acids which is the analyte of interest for any purpose. Thus, the oligonucleotide probe, via its selective binding capability, is employed to detect and identify individual gene fragments or nucleic acid sequences present in viruses, bacteria, and other cells serving as samples for scientific, research or medical interest.
In general, any DNA, RNA, or PNA sequential fragment (obtained from any source and regardless of whether the sequence is naturally occurring or synthetically prepared) must meet two essential criteria in order to be truly useful as an oligonucleotide probe. First, the oligonucleotide probe sequence must be as specific as possible for the intended complementary target sequence; and, preferably, bind exclusively with only the complementary target sequence with little or no cross-reaction. Secondly, the oligonucleotide probe must be able to distinguish among closely related nucleic acid base sequences having a substantial degree of homology as well as be able to bind selectively with varying types and sources of nucleic acid fragments having the complementary target sequence as part of its composition. Thus, the size or length of the oligonucleotide probe and the repetitive nature of or copy number for the complementary target sequence will meaningfully affect not only the specificity, but the sensitivity of the probe for detection purposes.
The technique employing an oligonucleotide probe for selective binding to a complementary target sequence is generally termed “hybridization”. However, the development of hybridization based assays for the identification of specific genes and gene expression products has been severely limited to date because of major difficulties in: (a) isolating highly specific nucleic acid sequences for use as oligonucleotide probes; (b) developing assay formats that are sufficiently rapid and simple in order to identify even one complementary target sequence in a fluid mixture containing many varieties of different single-stranded oligonucleotides in admixture; and (c) devising non-radioactive detection systems that provide a desired level of sensitivity. Thus, several types of DNA (or RNA, or PNA) hybridization assay formats have come into prevalent use.
Four hybridization assay formats are commonly employed today. Each of these hybridization detection formats suffers from relatively poor sensitivity, although various target sequence amplification techniques (such as PCR) have also been developed to reduce the severity of this problem. The four most commonly used types of hybridization assay formats are: the Southern blot technique; the dot or spot blot technique; in-situ hybridization; and sandwich hybridization assays. As with the selection of an appropriate oligonucleotide probe, the choice of a hybridization assay format often rests upon the degree of specificity and sensitivity that is required for the particular analysis; and upon the factors of speed, reliability, and ease of performance and interpretation of the assay result—which varies markedly among the different assay formats.
In Southern blot assays, specimen DNA is isolated and purified prior to restriction endonuclease digestion; followed by separation of the digestion products by electrophoresis on an agarose gel, denaturation of the DNA in the gel, and transfer of the denatured DNA fragments to a solid matrix such as a nitrocellulose membrane. The DNA bound to the solid matrix is then hybridized in the presence of radioactively labeled DNA targets to establish homology between the probe and target DNA. Hybridization of the targets to the probes is detected by autoradiography and often requires several days or weeks of exposure. This format is thus often too lengthy and cumbersome for routine or large-scale analyses of many specimens.
The dot-blot procedure also requires that specimen DNA be isolated and purified before being denatured and applied to a suitable solid matrix (such as nitrocellulose). Hybridization to the matrix-bound DNA is then performed using probe-specific targets. The hybridization of target DNA to the probe DNA is detected either by autoradiography or by visual inspection using non-radioactive detection procedures. The spot-blot assay format is similar except that specimens or specimen lysates are directly applied to the solid matrix without prior extraction of their DNA. Although this assay format allows many different samples to be processed at one time, these assays are often limited to high background noise that complicates the interpretation of results and is also subject to lengthy time of processing for each sample to be evaluated.
The in-situ hybridization technique intends that the DNA or RNA in the cells of a fixed tissue section or fixed culture cell be hybridized to DNA probes directly on a microscope slide. The results are determined by microscopy if non-radioactive detection systems are used and by autoradiography if radioisotopes are employed for the targets.
This assay format can detect the presence of only a few copies of the target DNA sequence to be hybridized. The conventional in-situ hybridization assay is not suitable for screening large numbers of specimens due to the need to separate and remove extraneous cellular materials from the sample prior to addition of the labeled target.
Lastly, the sandwich hybridization assay requires that at least two different specific probes hybridize to the target DNA of interest, rather than just one probe alone. In this format, the first probe (the capture sequence) is bound to a solid support and is allowed to bind (capture) the specimen DNA A second probe (the signaling probe) with a sequence that is adjacent or close to the capture sequence on the target DNA molecule is then allowed to hybridize to the support-bound target DNA. This signaling probe can be labeled with either radioactiv
Healey Brian G.
Walt David R.
Fredman Jeffrey
Silva Robin M.
Trustees of Tuft College
LandOfFree
Fiber optic biosensor for selectively detecting... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Fiber optic biosensor for selectively detecting..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Fiber optic biosensor for selectively detecting... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2942028