Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-04-08
2001-04-17
Arthur, Lisa B. (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S006120
Reexamination Certificate
active
06218151
ABSTRACT:
FIELD OF THE INVENTION
The present invention refers to the methods, reagents and kits for the amplification of target nucleic acid sequences. In particular, the present invention consists in a method for the amplification of nucleic acids by transcription, using displacement, and the detection of the amplification products obtained by this reaction.
DESCRIPTION OF THE RELATED ARTS
It is often necessary, in technologies relating to nucleic acids and to genetic material, to determine if a gene, a gene portion or a nucleotide sequence is present in a living organism, a cellular extract of this organism or a biological sample. Since any gene or gene portion is a specific sequence of nucleotide bases forming all or part of a nucleotide molecule, it is possible to directly search for the presence of a specific nucleotide sequence in a sample containing polynucleotides.
The usefulness of the search for specific nucleotide sequences is immense, especially for the detection of pathogenic organisms, the determination of the presence of alleles, the detection of the presence of lesions in a host genome and the detection of the presence of a specific mRNA or of the modification of a cellular host. Genetic diseases such as Huntington's disease, Duchenne's myopathy, phenylketonuria and &bgr;-thalassemia can be diagnosed by analysing DNA from the individual. Furthermore, the diagnosis or the identification of viruses, viroids, bacteria, fungi, protozoa or some other form of plant or animal life can be carried out by hybridization experiments with nucleic probes.
Various types of methods for the detection of nucleic acids are described in the literature. These methods, and particularly those which require the detection of polynucleotides, are based on the purine-pyrimidine pairing properties of the complementary strands of nucleic acids in the DNA—DNA, DNA-RNA and RNA—RNA complexes. This pairing process occurs by the establishment of hydrogen bonds between the adenosine-thymine (A—T) and guanosine-cytosine (G—C). bases of the double-stranded DNA; adenosine-uracil (A—U) base pairs can also be formed by hydrogen bonding in the 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 “nucleic acid hybridization” or simply “hybridization”.
In the few examples mentioned above, after identifying a sequence specific for an organism or for a disease, it is advisable to extract the nucleic acids from a sample, and to determine if this sequence is present. Numerous detection methods have been developed for this purpose.
While it is vital that one or more sequences specific for a disease or for an organism are identified, the nature of these sequences and the manner in which they were identified is of no particular importance for the implementation of the present invention. 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 a portion of the target nucleic acid to hybridize to the latter. The probe thus synthesized can be applied in a sample containing nucleic acids, and if the target sequence is present, the probe will hybridize in order to form a reaction product. In the absence of target sequence and by avoiding any non-specific hybridization phenomenon, no reaction product will be formed. If the synthesized probe is coupled to a detectable marker, the reaction product can be detected by measuring the quantity of marker present. Southern blotting (Southern E. M.,
J. Mol. Biol.,
98, 503 (1975)) or sandwich hybridization (Dunn A. R., Hassel J. A.,
Cell,
12, 23 (1977)) constitute examples where these methods are used.
The principal difficulty of this approach is, however, that it is not directly applicable to cases where the copy number of the target sequence present in a sample is low (that is to say less than 10
7
). Under these conditions, it is difficult to differentiate a significant signal, that is to say greater than the reaction background noise (that is to say to differentiate the specific binding of a probe onto its target sequence for the non-specific binding between the probe and a sequence different from the target sequence). One of the solutions to this problem consists of increasing the detection signal by an additional reaction. Consequently, 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 in a method of detection by nucleic probes. These stages can be classified into three categories: the amplification of target, of probe or of signal. The articles by Lewis (1992.
Genetic Engineering News
12:1-9) on the one hand, and by Abramson and Myers (1993.
Curr. Opin. Biotechnol.
4:41-47) on the other hand, constitute good general reviews of these methods.
The amplification of target consists of multiplying, in specific manner, a nucleic acid fragment present in a sample. It makes it possible to considerably increase the copy number of a target nucleic sequence to be detected.
The most widely known method is the Polymerase Chain Reaction (called PCR), a target amplification technique which is based on the repetition of cycles of DNA synthesis in vitro by extension of nucleotide primers hybridized with the target sequence (Saiki et al., 1985.
Science
230:1350-1354; U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159; European Patent No. 0,201,184). Briefly, two nucleotide primers each complementary to a sequence of one of the two strands of the target DNA are synthesized. Deoxyribonucleoside triphosphates are added in excess to the reaction medium in the presence of a thermostable DNA-dependent DNA polymerase (Tag polymerase). If the target DNA is present in the sample, the primers hybridize with their specific sites and the polymerase extends the 3′ end of these primers by adding successively nucleotides complementary to the target. By carrying out successive cycles of rise and fall in temperature, the extended primers separate from the target and can, like the original target, bind the nucleotide primers in excess. By repeating the process (from 20 to 40 times, an exponential accumulation of the target sequence between the two primers is obtained.
Another method using temperature cycles is described in European Patent No. 0,320,308 and is called Ligase Chain Reaction (termed LCR). Two adjacent oligonucleotide probes, as well as two others which are complementary to them are added to the reaction medium in excess, in the presence of a DNA ligase. In the presence of the target DNA, each oligonucleotide hybridizes with its complementary sequence and the ligase can link the two adjacently hybridized probes. By a succession of temperature cycles, like in PCR, and by the use of a thermostable ligase (Barany, 1991.
Proc. Natl. Acad. Sci. USA
88:189-193), the linked probes separate from the target and can in turn serve as target sequence for the probes in excess.
The Repair Chain Reaction method (termed RCR) is an amplification method similar to LCR (Patent Application No. WO 90/01069). It is a hybrid method between PCR and LCR. It uses two oligonucleotide probes complementary to the target and two primers in excess in the reaction medium, in the presence of a thermostable DNA ligase and a thermostable DNA polymerase. In the presence of target DNA, the oligonucleotides hybridize with their target sequence and a gap of a few bases separates the end of each primer from the adjacent oligonucleotide probe. The polymerase fills this gap and also makes possible the action of the ligase which links the extended primer to the adjacent probe, thus mimicking the natural processes of DNA repair. By a succession of temperature cycles, as in PCR and LCR, the extended primers linked to the oligonucleotide probes can in turn serve as target for the primers and the probes in excess.
Other target amplification methods exist which are
Cleuziat Philippe
Guillou-Bonnici Francoise
Levasseur Pierre
Mallet Francois
Arthur Lisa B.
Bio Merieux
Oliff & Berridg,e PLC
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