Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-01-08
2000-03-07
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 231, 536 243, 935 76, 935 77, 935 78, C12Q 168, C07H 2102, C07H 2104, C12N 1500
Patent
active
060338539
DESCRIPTION:
BRIEF SUMMARY
The subject of the present invention is a reagent and a process for the detection of a nucleotide sequence in a sample.
It is often necessary to determine if a gene, a gene portion or a specific nucleotide sequence is present in a living organism, in a cellular extract or in biological samples.
The search for specific nucleotide sequences is used in particular for the detection of pathogenic organisms, the determination of the presence of alleles or the detection of the presence of lesions in a genome. Genetic diseases such as Huntington's disease, Duchenne's myopathy, phenylketonuria and .beta.-thalassaemia can be diagnosed through the analysis of DNA from individuals. Furthermore, the diagnosis or the identification of viruses, of viroids, of bacteria, of fungi or of parasites can be carried out by hybridization experiments with nucleic probes.
Various types of methods of detection of nucleic acids are described in the literature. These methods are based on the purine-pyrimidine pairing properties of the complementary strands of nucleic acids in DNA--DNA, DNA-RNA and RNA--RNA duplexes. This pairing process occurs through the establishment of hydrogen bonds between the adenosine-thymine (A-T) and quanosine-cytosine (G-C) bases of the double-stranded DNA. Adenosine-uracil (A-U) base pairs can also be formed through hydrogen bonds in DNA-RNA or RNA--RNA duplexes. The pairing of nucleic acid strands for the determination of the presence or of the absence of a given nucleic acid molecule is commonly called "hybridization of nucleic acids" or simply "hybridization".
On the basis of the properties of nucleic acids, techniques have been developed which make it possible to detect and quantify, in a sample to be analysed, a nucleic acid called target. These techniques, which are well known, can be divided into two main groups: the so-called direct detection techniques such as that of the so-called SOUTHERN technique and the so-called "Dot-blot" technique for the detection of DNA or the NORTHERN technique for the detection of RNA, and the so-called indirect techniques such as the sandwich or "Reverse-Dot" technique.
One of the principal difficulties encountered during the development of a test for detecting a target nucleotide sequence of interest is the sensitivity threshold of the hybridization methods, and various methods have been described in order to increase the power of detection of these hybridization techniques. These so-called "amplification" methods can be used at various stages of a detection process using nucleic probes. Two categories can be distinguished: amplification of a target or of a signal.
The techniques for amplification of a target are known. One disadvantage of these techniques lies in the difficulty of quantifying the nucleic target of interest after the amplification step.
Other approaches, relating to the amplification of a signal, have been described. Thus, U.S. Pat. No. 4,731,325 and EP-0,225,807 describe techniques using a plurality of detection probes which can hybridize to the target. In many cases (necessity to differentiate between related species in bacteriology or detection of genetic diseases), it is not possible to use this technique because only one specific sequence on the target is capable of being used to hybridize a detection probe.
Some techniques described consist in increasing the number of markers, that is to say of molecules capable of generating a signal, directly or indirectly, on the detection probe. The marker may be in particular a biotin, a fluorophore, an enzyme or a radioactive group. The detection probe is grafted onto a polymer, which may be of a nucleotide nature, onto which are attached, most often through covalent bonding, a number of markers greater than two (see for example U.S. Pat. No. 4,687,732, EP-0,373,956, WO-88/02784, WO-90/00622, EP-0,292,128, WO-89/03849 and EP-0,173,339).
One of the disadvantages of these techniques lies in the need to carry out controlled coupling between the probe and the number of markers. This double coupling is not easy
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Charles Marie-Helene
Delair Thierry
Elaissari Abdelhamid
Mandrand Bernard
Bio Merieux
Jones W. Gary
Whisenant Ethan
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