Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1985-11-22
1989-02-28
Warren, Charles F.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
935 78, 536 27, 435803, 4351723, 435 29, C12Q 168, C07H 2100, C12N 1500
Patent
active
048085197
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates to the specific detection of DNA of a specific base sequence. In particular, it relates to the construction of a DNA molecule, termed a "probe-vector", which is complementary to the DNA sequence that one wishes to detect, called the "target" sequence, and which will transform bacteria at high efficiency if and only if it has hybridized with the target sequence.
Deoxyribonucleic acid, or DNA, is a long linear polymer of units called nucleotides. Each nucleotide contains any one of the four nitrogeneous bases adenine (A), guanine (G), cytosine (C), and thymine (T). The sequence of bases in an organism's DNA specifies the genetic characteristics of the organism. Most of the individual organisms belonging to a species share most of their respective DNA sequences in common. Accordingly it is possible to identify DNA sequences which all or most of the individual organisms of a species contain but which do not exist in organisms outside the species. Such a DNA is characteristic of the species, and is in a sense "diagnostic" of it.
The ability to detect and identify particular species has application in the diagnosis of infectious diseases. Various pathogens, for example, viruses, bacteria, fungi, and protozoa, can be detected and identified by detecting particular DNA sequence in clinical specimens by this invention. Further genetic characteristics of an infecting organism which affect the pathogenicity or resistance to therapeutic agents (for example antibiotic resistance) can also be detected and identified by this invention.
Within a species, individual organisms exhibit genetic differences from one another. In some cases these differences are manifested as inherited diseases, such as sickle cell anemia in man. These differences can be detected as differences in the base sequence of the DNA of the various organisms. Other diseases such as diabetes and heart disease have genetically determined predispositions which can be identified by characteristic variations in the DNA sequence of the individual. This invention can be applied to detect and identify these variations, and thereby, the genetic predispositions they indicate.
Rearrangements of genomic DNA can result in sequences which were formerly far away from each other being brought into close proximity. Such genetic transpositions occur during development of the immune system, and are implicated in the etiology of some cancers. The probe-vector of this invention requires close linkage between two target sequences for detection of those sequences. Thus suitable probe-vectors can be used to detect rearranged sequences resulting from genetic transpositions.
Since the characteristic DNA sequence one wishes to identify may (is likely to) be found in the presence of a vast abundance of DNA of different sequence it is necessary that its method of detection be highly specific. Further, since little DNA of the characteristic sequence may be available for analysis, a method of high sensitivity is also desirable.
DNA possesses a fundamental property called base complementarity. In nature DNA ordinarily exists in the form of pairs of anti-parallel strands, the bases on each strand projecting from that strand toward the opposite strand. The base adenine (A) on one strand will always be apposed to the base thymine (T) on the other strand, and the base guanine (G) will be apposed to the base cytosine (C). The bases are held in apposition by their ability to hydrogen bond in this specific way. Though each individual bond is relatively weak, the net effect of many adjacent hydrogen bonded bases, together with base stacking effects, is a stable joining of the two complementary strands. These bonds can be broken by treatments such as high pH or high temperature, and these conditions result in the dissociation, or "denaturation", of the two strands. If the DNA is then placed in conditions which make hydrogen bonding of the bases thermodynamically favorable, the DNA strands will anneal, or "hybridize", and reform the original d
REFERENCES:
patent: 4394443 (1983-07-01), Weissman et al.
patent: 4666839 (1987-05-01), Souza
Chem. Abst., vol. 104, No. 11, issued Mar. 17, 1986, p. 173, abst. 83081s, Hartley, J. et al., "Detecting Nucleic Acid Sequences".
Supplement European Search Report, (Mar. 2, 1988).
Berninger Mark S.
Hartley James L.
Jay Jeremy M.
Life Technologies Inc.
Warren Charles F.
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