Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-08
2001-05-29
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C422S068100
Reexamination Certificate
active
06238866
ABSTRACT:
GOVERNMENT INTEREST
The invention described herein may be manufactured, licensed, and used by or for the U.S. Government.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is generally related to the field of detecting and identifying genetic materials. such as nucleic acid molecules of interest, using a nucleic acid detector. The nucleic acid detector according to the invention is constructed from double stranded hybridization probes that are immobilized on a support, to form a DNA detector. The DNA detector is formed into an array of varied binding specificities that is capable of detecting multiple genetic loci. The probes are oligonucleotides each comprising a hypervariable number of tandem repeats (“VNTR”) that are anchored at one terminus to a support and at the other terminus to a reporter moiety.
2. Description of the Related Art
Previously, the best method known for detecting and identifying a nucleic acid analyte (e.g., an unknown sample) has been so-called DNA fingerprinting, which relies on a comparison of the electrophoretic migration of restriction fragments of an unknown nucleic acid analyte to the electrophoretic migration pattern of a known genomic sample subjected to identical restriction treatment. This process is typically, but not exclusively, employed to identify or detect deoxyribonucleic acid (“DNA”). Variations on this process are known, including the use of specific hybridization probes to enhance accuracy by confirming that migration bands are homologous to specific nucleic acid sequences of interest. This is possible because restriction enzymes cleave DNA at specific loci, which will vary (i.e., exhibit polymorphism) with each genome. Thus, when DNA gel banding techniques where developed, it seemed possible that the technique would provide unique and unequivocal comparisons and identification between genomic samples.
DNA fingerprinting has been relied upon to analyze forensic evidence, for example, to obtain evidence of the identity of genetic material for criminal or paternity proceedings. The technique is also used to identify human remains or to determine species relatedness. DNA fingerprinting using VNTR loci complementary probes is useful not only in a forensic laboratory setting to provide individual identification and paternity testing, but also has been extensively used for the investigation of taxonomic relationship among fish, birds, plants, wild and domestic animals. In addition, it has also been used for clarifying genetic relationship among related species, for discriminating pathogens from non-pathogens, and for determining the effect of environmental factors on evolutionary dynamics and speciation of microorganisms.
Of course, as the artisan will appreciate, the accuracy of the DNA fragment size-sharing principle has been the subject of criticism e.g., in numerous legal cases, because similarities in the electrophoretic mobility, e.g., size, of restriction fragments on DNA electrophoretic profiles does not unequivocally establish the full characteristics of the alleles.
Therefore, greater accuracy was required before DNA gel banding techniques would be accepted as an accurate means of genetic profiling. While the classical genetic markers. such as the single-locus probe (SLP), have been employed in an attempt to provide improved identification. these usually give little information about the individualization of species due to the very large number of SLP's necessary to determine the exact relationship between species. The use of multiple SLPs for identification purposes has been known as “multiplexing.”
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In 1985, geneticist Alec Jeffreys (Jeffreys, et al., 1985, Nature 314:67-73) partially solved the accuracy problem by applying certain specific hybridization probes to electrophoresis patterns produced by DNA restriction fragments. Since then. while DNA profiling has become an accepted scientific and forensic tool, a great deal of public controversy has continued to be generated by these methods, particularly when used for forensic identifications.
The method described by Jeffreys et al., supra, was based on DNA gel banding techniques with the added feature of detecting polymorphisms in minisatellite DNA. The term “satellite” was originally derived from the observation that DNA isolated from eukaryotes under buoyant density gradient ultracentrifugation, shows extra peaks beside the major DNA bands (Bretton. R J et al., 1968
. Science
161: 529-540). Satellites are relatively large chromosomal structures which contain millions of repetitive sequences. Similar repetitive sequences were later found to be almost universally present in the genomic structures of most eukaryotes, as well as in genome viruses. These sequences were called “minisatellites”, “midisatellites”, and “microsatellites” because of their limited degrees of repetition, i.e., small size (Jeffreys et al., 1985, supra) relative to “satellites”, which contain millions of repetitive sequences.
For example, the term, “minisatellite” is applied to any of a class of dispersed arrays of short (e.g., 10-50 bp) tandem direct repeat motifs that contain variants of a common core sequence (e.g., 10-15 bp). The majority of minisatellites are distributed at the terminal ends of genomes. Although their exact functions are not clearly known these terminal repeats may play a significant role in replication control of genes. The major difference of TR's from classical genetic markers is in the hypervariable number of the tandem repeats (VNTR). The VNTR therefore provides extremely useful information about relatedness and individualization of species and other types of nucleic acid analytes.
The human-derived minisatellites such as 33.6,33.15, MS1, CMM1O 1YNH24, EFDS2, TBQ7, MS43 and JE46 are commonly used to prepare hybridization probes for forensic testing, for example, to provide individual identification and paternity testing. These minisatellites can also hybridize to DNA isolated from avians, plants, fish, and other mammals
3-6
, thus indicating the presence of common genetic structures in these living organisms.
The multiloci human probes 33.6, 33.15, and MS1 have been used for fingerprinting of DNA isolated from pigs, mice, and common marmosets,
3
for characterization of genetic relationship between breedings of poultry, and for studying a population of foxes in the California Channel Islands.
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Application of the multilocus fingerprinting probe (MLP) 33.15 also revealed genetic profiles in species and strains of Leishmania and
Trypanosoma cruzi.
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Fingerprinting data can permit the construction of pedigrees that reflect the population history and the geographical distance of different species and strains of the parasites.
Another type of abundantly distributed repetitive sequences are the microsatellites, which have an even smaller degree of repetition than that of the minisatellites. They are distinguishable from minisatellites by having repetition within the repetitive units. The majority of minisatellites are usually distributed at the terminal ends of genes, while microsatellites are widespread along the chromosomes. Microsatellites used extensively for DNA-fingerprinting have the general structural characteristics of (CA)8, (CT)8, (CAC)5, (GAC)5, (GACA)4, (GATA)4, and the like. They are abundantly distributed in genomic structures of living organisms. Dinucleotide repeats, particularly CA/GT repeats, are very abundant and polymorphic. In other words, they are extremely variable in number of the repeat units.
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Using (GTG), (GACA) or phage M13 core sequence as either hybridization probes or primers in combination with restriction enzymes with a recognition site of 6 base pairs (bp), over 70 species representing 18 genera filamentous fungi and 5 genera of yeasts were fingerprinted, and their DNA banding patterns and taxonomic relationship were clearly identified.
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These ubiquitously interspersed, tandemly repetitive sequences with a total number of bases ranging from 2 to 6 are highly polymorphic, rendering these simple short tandemly
Wick Charles H.
Yeh Homer R.
Biffoni Ulysses John
Marschel Ardin H.
Ranucci Vincent J.
The United States of America as represented by the Secretary of
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