Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-02-02
2003-09-09
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091100, C435S006120
Reexamination Certificate
active
06617138
ABSTRACT:
The present invention relates to nucleic acid detection methods, in particular to quantitative nucleic acid detection methods.
Detection, especially quantitative detection of a particular nucleic acid sequence, as an indication of the presence of an organism e.g. a pathogen in a clinical sample or a contaminant in a food or environmental sample, such as toxin-producing cyanobacteria in water sources, or of mRNA to show a change in transcription levels, is a valuable microbiological tool. In addition, in the diagnostic or forensic use of nucleic acid analysis or in the study of polymorphisms, full sequencing of the target nucleic acid may be unnecessary where the detection of a single base variation or mismatch is sufficient to give a positive identification. Such a single base variation or mismatch may, for example, arise from allelic variation or polymorphism, a point mutation, or any deletion or insertion of genetic material where the detection of a single abnormal or species specific base will give the required information.
Through our work in the development of methods for the detection of bacteria in water, we have developed a new nucleic acid detection method suitable for a wide variety of applications in the environmental, agricultural, food, veterinary, health and medical fields and indeed as a general tool in molecular biology.
There are a number of techniques available for the analysis of nucleic acids including the manufacture of synthetic oligonucleotide probes, particularly labelled probes, for hybridisation to target sequences; in vitro amplification of target nucleic acid sequences by PCR and other related amplification methods and (automated) direct DNA sequencing. These have led to the development of novel approaches for the detection and characterisation of nucleic acids in environmental monitoring (Bej, A. K. and Mahbubani, M. H. [1994] in PCR Technology: Current Innovations p 327-339 and Bowman, J. P. and Sayler, G. S. [1996] in Molecular approaches to environmental microbiology p 63-97). There are three main strategies for quantification of amplified DNA; i.e. size separation by electrophoresis, hybridization to capture probes, and real-time detection. The problems with the gel electrophoresis method are the detection of multiple targets in a single reaction, and interpretation of the results. Size separation detection of multiplex amplifications is also difficult to achieve because the amplification ratios of amplicons with different sizes are dependent on DNA quality.
The capture probe assay is based on hybridization of the entire amplified fragments. Evidently, this assay is not suitable for separation and quantification of homologous amplicons, e.g. products of competitive amplifications. The different amplicons will form sandwich hybridizations at the homologous sites, leading to the capture of both target and non-target fragments, even if the capture site is discriminating.
The ABI PRISM™ 7700 Sequence Detection System (Perkin Elmer, Foster City, Calif.) provides real-time quantitative PCR amplification. However, multiplex assays are limited by the number of fluorochromes available and their overlapping fluorescent spectra.
It is always an aim in nucleic acid detection methods to increase their specificity, ie. to reduce non-specific binding or detection of a non-specific background signal. A separate but associated goal is to increase the sensitivity of the detection method, ie. to allow measurement of very small amounts of target nucleic acid.
It would also represent a significant advantage if the method allowed the detection and quantification of several, preferably a large number such as 10 or more, polymorphic sites in a single reaction e.g. allowing the detection and quantification of several different target organisms in a single multiplex assay.
We have developed a convenient detection method which provides good sensitivity and specificity and the ability to detect and quantitate a large number of polymorphic sites in a single reaction. In particular, the methods represent significant advantages over those employing agarose gel electrophoresis or direct detection of the amplified DNA in the quantification.
The invention thus provides a method of detecting a target nucleotide sequence in a nucleic acid molecule, which comprises:
(a) binding of an oligonucleotide probe to said nucleic acid molecule;
(b) selective labelling of the bound oligonucleotide probe in the presence of said target nucleotide sequence;
(c) hybridisation of the labelled oligonucleotide to a complementary sequence; and
(d) subsequent detection of the label.
The method of the invention can be used in the detection of all target nucleotide sequences, such as DNA sequences, particularly DNA resulting from PCR amplification cycles. The DNA may be native or cDNA formed from mRNA by reverse transcriptase. The DNA may be single or double stranded, linear or circular. The target nucleic acid may be RNA, e.g. mRNA or in particular ribosomal RNA which is present in a cell in multiple copies, for example 3,000-20,000 copies per cell.
In the context of the present invention, the term “nucleotide sequence” may refer to a ‘sequence’ of only one nucleotide in length, where the nucleic acid of interest differs by only one nucleotide from other (non-target) sequences which it is not desired to detect, such as in the case of detection of a point mutation or polymorphism. More usually, the nucleotide sequence to be detected is characteristic of a particular nucleic acid or group of nucleic acids or of a particular organism or group of organisms e.g. a species, where it is desired to detect the presence of that target (e.g. the nucleic acid/or organism) in a sample containing a number of different molecules or organisms, such as in the detection of particular bacteria e.g. in a clinical or environmental sample.
The oligonucleotide probe may comprise 5 to 50, preferably 10 to 40, more preferably 20 to 30 nucleotides. The probe is selected to bind to the target nucleic acid, namely the nucleic acid molecule containing the target nucleotide sequence. The oligonucleotide is conveniently sufficiently large to provide appropriate hybridisation to the target nucleic acid, yet still reasonably short in order to avoid unnecessary chemical synthesis. Methods for oligonucleotide production are standard in the art.
As discussed above, the target nucleic acid may be DNA, cDNA or RNA. The oligonucleotide probe is designed to bind to a target region in the nucleic acid molecule containing the target nucleotide sequence. “Target region” is used herein to refer to the sequence of nucleic acid which binds to the oligonucleotide probe. This target region may be in the form of a “signature sequence” which characterises a particular nucleic acid or group of nucleic acids or organisms etc. or may be the section of nucleic acid immediately preceding a single base position of interest, if a polymorphism, point mutation, insertion or deletion is to be detected.
It is of course desirable that the target region be absent from any non-target molecules in the sample in order to introduce selectivity between target and non-target nucleotide sequences. This specificity of binding may be achieved by incorporating into the probe regions of complementarity or substantial complementarity to the desired target region and/or mismatches to non-target sequences. The region of complementarity may be present at terminal or internal segments of the probe, or both.
Conventionally, nucleic acid detection methods have involved labelling of the oligonucleotide probe prior to hybridisation to the target nucleic acid sequence. However, in our method, labelling of the probe is dependent upon the sequence of the nucleic acid molecule to which the probe is bound. In other words labelling of the probe is template-sequence dependent (i.e. sequence-specific)—incorporation of the label will is only occur in the presence of a desired or selected “sequence” in the target molecule, which may be in the target region or in the targ
Jakobsen Kjetill Sigurd
Rudi Knut
Dorsey & Whitney LLP
Einsmann Juliet
Genpoint AS
LandOfFree
Nucleic acid detection method does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleic acid detection method, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid detection method will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3027848