Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-07
2002-08-27
Benzion, Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C436S094000, C536S022100, C536S025320, C536S026600, C536S026740
Reexamination Certificate
active
06440671
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for analyzing DNA sequences and more particularly this invention relates to a method for using sequencing by hybridization with oligonucleotides associated with polyacrylamide matrices, including continuous/contiguous stacking hybridization methods, to detect disease-associated alleles.
2. Background of the Invention
Present techniques for determining the existence of disease-associated alleles in patient DNA are complex, inefficient and somewhat time consuming. This is due to the fact that technologies applied to mutation location stem from complex and other error-prone base sequencing procedures. For example, one multi-step DNA sequencing approach, the Maxam and Gilbert method, involves first labeling DNA, and then splitting the DNA with a chemical, designed to alter a specific base, to produce a set of labeled fragments. The process is repeated by cleaving additional DNA with other chemicals specific for altering different bases, to produce additional sets of labeled fragments. The multiple fragment sets then must be run side-by-side in electrophoresis gels to determine base sequences.
Another sequencing method, the dideoxy procedure, based on Sanger, et al.
Proc. Natl. Acad. Sci. USA
74, 5463-7 (1977) first requires the combination of a chain terminator as a limiting reagent, and then the use of polymerase to generate various length molecules, said molecules later to be compared on a gel. The accompanying lengthy electrophoresis procedures further detracts from the utility of this method as a fast and efficient diagnostic tool.
A more recently developed sequencing strategy involves sequencing by hybridization on oligonucleotide microchips, or matrices, (SHOM) whereby DNA is hybridized with a complete set of oligonucleotides, which are first immobilized at fixed positions on a glass plate or polyacrylamide gel matrix. There are drawbacks to this technique, however. For instance, given that short nucleotide sequences are repeated rather frequently in long DNA molecules, the sequencing of lengthy genome strings is not feasible via SHOM.
Furthermore, the procedures for manufacturing sequencing microchips with the required, large number of immobilized oligonucleotides is not perfected. For example, if immobilized octamers are utilized to determine the positions of each of the four bases in genomic DNA, then 4
8
or 65,536 such octamers need to be fabricated and subsequently immobilized on the gel. Also, hybridization with short oligonucleotides is affected by hairpin structures in DNA.
Yet another disadvantage in using SHOM is its ineffectiveness in discriminating perfect DNA-oligomer duplexes from mismatched ones, particularly mismatched duplexes at terminal positions. Such terminal mismatches are harder to discriminate than internal mismatches.
In a variation of SHOM, sequencing of DNA strings is facilitated via a contiguous stacking hybridization (CSH) approach, whereby the microchip, comprising a gel embedded with immobilized oligomer such as an octamer (8-mer), is hybridized first with DNA and then with a fluorescently labeled oligomer such as a pentamer (5-mer). The resulting, contiguous 13 base-long oligomer (the 5-mer in a juxtaposed position to the immobilized 8-mer) thus formed acts as a probe to the DNA region.
The efficiency of CSH is due to a more stable probe being formed when the immobilized oligomer is positioned side by side with the mobilized oligomer. This extended complimentary probe therefore results in a more stable duplex between the probe and target DNA.
As with SHOM, however, there are drawbacks with CSH. First, in addition to the 65,536 immobilized oligomers already required to produce the immobilized oligo fraction in the gel matrix (discussed supra), the number of mobile oligomers (i.e. mobile pentamers) necessary to completely read the subject DNA via CSH is also formidable. When mobile pentamers are used, for example, given the possibility of any one of four bases at any one base position on the pentamer, all variations of the pentamer (4
5
=1,024) must be produced and hybridized with the chip. Furthermore, the microchip, containing the duplexed DNA must be contacted with all the 1,024 pentamers in separate hybridization procedures (i.e. performing 1,024 additional hybridization rounds) to fully sequence the subject DNA.
Hybridization of filter-immobilized DNA with oligonucleotides in solution also has been suggested for mutation detection. However, this approach is too cumbersome for screening all possible base changes in some genes. For example, in the case of &bgr;-thalassemia, the number of changes exceeds 100.
A need exists in the art to provide an efficient method for diagnosing disease by detecting multiple mutation sequences in patient DNA. Such a method must incorporate a minimal number of oligonucleotides and utilize a minimal number of hybridization steps. The method also must be of sufficient efficiency so as to effectively discriminate perfect duplexes from imperfect ones.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for detecting multiple DNA base mutations, which are specific for certain diseases, that overcomes the disadvantages of the prior art.
Yet another object of the present invention is to provide a method to sequence target DNA by hybridizing the DNA first to oligonucleotide microchips and then subjecting the resulting DNA-oligo duplex to mobile oligonucleotides. A feature of the invented method is using a minimal number of mobile oligonucleotides to extend the sequences of immobilized nucleotides which are complementary to disease-associated alleles. An advantage of the invented method is enhanced detection of the DNA-oligonucleotide duplex.
Still another object of the present invention is to provide a procedure for more accurately detecting the presence of disease-associated DNA mutations. A feature of the invention is the use of universal bases or a mixture of all four bases in oligonucleotide probe sequences. An advantage of the method is producing a more sensitive method for discriminating perfect duplexes from mis-matched duplexes in SHOM procedures. Another advantage is to increase the efficiency of CSH by reducing the number of mobile oligomers and hybridization rounds.
Another object of the invented method is to provide a procedure, incorporating a minimum number of stacking hybridization steps, that can accurately determine the existence of disease-associated DNA mutations. A feature of the method is the simultaneous hybridization of patient DNA, first duplexed with immobilized DNA, with mobile oligonucleotide probes, each of said probes containing a different fluorochrome. An advantage of the invented method is to decrease the number of hybridization steps, thereby expediting the process of mutation detection.
Yet another object of the present invention is to provide a method for sequencing DNA and RNA molecules with elongated, immobilized probes. A feature of the invention is the ligating together of probes after their juxtaposition to each other on an immobilization substrate. An advantage of the invention is the ability to sequence-test long DNA and RNA molecules containing repeat regions. An additional advantage is the use of the invented method to simplify the sequencing of similar genes and genomes.
Still another object of the present invention is to provide a diagnostic method for detecting disease. A feature of the invention is the covalent extension of probes to a target DNA or RNA molecule. An advantage of the invention is the use of the probes as a diagnostic tool to determine the extent of the existence of repeat sequences in the target molecule, the existence of which is often proportional to severity of disease coded by the molecules.
In brief, the objects and advantages of the present invention are achieved by a method for detecting disease associated alleles in patient genetic material comprising immobilizing a first group of oligonucleotide molecules of a prede
Barski Victor Evgenievich
Dubiley Svetlana Alekseevna
Kirillov Eugene Vladislavovich
Mirzabekov Andrei Darievich
Parinov Sergei Valeryevich
Benzion Gary
Cherskov & Flaynik
The University of Chicago
Tung Joyce
LandOfFree
Use of continuous/contiguous stacking hybridization as a... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of continuous/contiguous stacking hybridization as a..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of continuous/contiguous stacking hybridization as a... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2902690