Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1997-05-16
1998-10-20
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 435 911, 536 2433, C12Q 168, C12P 1934, C07H 2104
Patent
active
058245170
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to the amplification of nucleic acid sequences. In particular, the present invention relates to a process of nucleic acid amplification as well as the reagents to be used in this process.
DESCRIPTION OF RELATED ARTS
In nucleic acid and genetic material technologies, it is often necessary to determine whether a gene, a part of a gene, or a nucleotide sequence is present in a living organism, a cellular extract of this organism, or a biological sample. Since any gene or part of a gene is characterized by a specific sequence of nucleotide bases, one need only search directly for the presence of all or part of said specific sequence in a sample containing a mixture of polynucleotides.
There is enormous interest in this search for specific polynucleotide sequences, particularly in detection of pathogenic organisms, determination of the presence of alleles, detection of the presence of lesions in a host genome, or detection of the presence of a particular RNA or modification of a cell host. Genetic diseases such as Huntington's disease, Duchenne's disease, phenylketonuria, and .beta. thalassemia can thus be diagnosed by analyzing nucleic acids from the individual. Also it is possible to diagnose or identify viruses, viroids, bacteria, fungi, protozoans, or any other form of plant or animal life by tests employing nucleic probes.
In the few examples cited above, once a specific sequence of an organism or a disease has been identified, the nucleic acids should be extracted from a sample and a determination should be made as to whether this sequence is present.
Various methods of nucleic acid detection have been described in the literature. These methods are based on the properties of purine-pyrimidine pairing of complementary nucleic acid strands in DNA-DNA, DNA-RNA, and RNA-RNA duplexes. This pairing process is effected by establishing hydrogen bonds between the adenine-thymine (A-T) and guanine-cytosine (G-C) bases of double-stranded DNA; adenine-uracil (A-U) base pairs can also form by hydrogen bonding in DNA-RNA or RNA-RNA duplexes. The pairing of nucleic acid strands for determining the presence or absence of a given nucleic acid molecule is commonly called "nucleic acid hybridization" or simply "hybridization."
The most direct method for detecting the presence of a target sequence in a nucleic acid sample is to obtain a "probe" whose sequence is sufficiently complementary to part of the target nucleic acid to hybridize therewith. The probe thus synthesized can be applied in a sample containing nucleic acids. If the target sequence is present, the probe will form a hybridization product with the target. In the absence of a target sequence, no hybridization product will form. If the synthesized probe is coupled with a detectable marker, the hybridization product can be detected by measuring the quantity of marker present. A transfer of the Southern (Southern, E. M., J. Mol. Biol., 98, 503 (1975)) or Northern type or the dot-blot or hybridization sandwich technique (Dunn, A. R. and Hassel J. A., Cell, 12, 23, (1977)) are examples in which these methods are used.
The main difficulty in this approach, however, is that it is not directly applicable to cases where the number of copies of the target sequence present in a sample is small, less than approximately 10.sup.7. Under these conditions it is difficult to distinguish a significant signal, exceeding the background noise of the reaction (namely to distinguish specific attachment of a probe to its target sequence from nonspecific attachment of the probe to a sequence different from the target sequence). One of the solutions to this problem consists of augmenting the detection signal by a preliminary technique designed to specifically and considerably increase the number of copies of a target nucleic acid fragment if it is present in the sample. A technique of this type is currently called an amplification technique.
The articles by Lewis (1992, Genetic Engineering News 12: 1-9) and Abramson and Myers (1993
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Proceedings of the National Academy of Sciences of USA, vol. 89, No. 1, Jan. 1, 1992, pp. 392-396, Terance Walker, "Isothermal in vitro amplification of DNA by a restruction enzyme/DNA polyermase system".
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Cleuziat Philippe
Mandrand Bernard
Bio Merieux
Horlick Kenneth R.
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