Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-07-19
2002-05-21
Whisenant, Ethan C. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C436S094000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06391593
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the introduction of destabilizing moieties into oligonucleotide probes for the improvement of nucleic acid amplification processes, methods comprising the use of such oligonucleotide and to kits for performing nucleic acid amplification processes comprising such oligonucleotide probes. The present invention is particularly concerned with amplification of hybridised modified nucleic acid probes such that sensitivity and specificity of the reaction is increased.
BACKGROUND OF THE INVENTION
All publications mentioned in the present specification are herein incorporated by reference.
A number of nucleic acid amplification processes are cited in the literature and disclosed in published European and PCT patent applications. One such process known as polymerase chain reaction (PCR) is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202. The PCR process consists of nucleic acid primers that anneal to opposite strands of a DNA duplex; these primers are extended using thermostable DNA polymerase in the presence of nucleotide triphosphates to yield two duplex copies of the original nucleic acid sequence. Successive cycles of denaturation, annealing and extension are undertaken to further amplify copies of the original nucleic acid sequence. This method has its drawbacks including the need for adjusting reaction temperatures alternately between intermediate (e.g. 50° C.-55° C.) and high (e.g. 90° C.-95° C.) temperatures involving repeated thermal cycling. Also the time scale required for multiple cycles of large temperature transitions to achieve amplification of a nucleic acid sequence and the occurrence of sequence errors in the amplified copies of the nucleic acid sequence is a major disadvantage as errors occur during multiple copying of long sequence tracts. Additionally, detection of the amplified nucleic acid sequence generally requires further processes e.g. agarose gel electrophoresis.
Alternative nucleic acid amplification processes are disclosed WO 88/10315 (Siska Diagnostics). EP 329,822 (Cangene) and 373,960 (Siska Diagnostics). U.S. Pat. No. 5,554,516 (Gen-Probe Inc.), and WO 89/1050 & 88/10315 assigned to Burg et al. and Gingeras et al., respectively. These amplification processes describe a cycling reaction comprising of alternate DNA and RNA synthesis. This alternate RNA/DNA synthesis is achieved principally through the annealing of oligonucleotides adjacent to a specific DNA sequence whereby these oligonucleotides comprise a transcriptional promoter. The RNA copies of the specific sequence so produced, or alternatively an input sample comprising a specific RNA sequence (U.S. Pat. No. 5,554,516), are then copied as DNA strands using a nucleic acid primer and the RNA from the resulting DNA:RNA hybrid is either removed by denaturation (WO 88/10315) or removed with RNase H (EP 329822, EP 373960 & U.S. Pat. No. 5,554,516). The annealing of oligonucleotides forming a transcription promoter is then repeated in order to repeat RNA production.
Amplification is thus achieved principally through the use of efficient RNA polymerases to produce an excess of RNA copies over DNA templates. The RNase version of this method has great advantages over PCR in that amplification can potentially be achieved at a single temperature (i.e. isothermally). Additionally, a much greater level of amplification can be achieved than for PCR i.e. a doubling of DNA copies per cycle for PCR, compared to 10-100 RNA copies using T7 RNA polymerase. A disadvantage associated with the DNA:RNA cycling method described in EP 329822 is that it requires test nucleic acid with discrete ends for the annealing of oligonucleotides to create the transcriptional promoter. This poses difficulties in detection of, for example, specific genes in long DNA molecules. Further disadvantages of this method are that at least three enzymes are required to undertake the DNA:RNA cycling with potentially deleterious consequences for stability, cost and reproducibility; and that one or more further processes are often required (e.g. gel electrophoresis) for detection of the amplified nucleic acid sequence.
The processes described above all refer to methods whereby a specific nucleic acid region is directly copied and these nucleic acid copies are further copied to achieve amplification. The variability between various nucleic acid sequences is such that the rates of amplification between different sequences by the same process are likely to differ thus presenting problems for example in the quantitation of the original amount of specific nucleic acid.
The processes listed above have a number of disadvantages in the amplification of their target nucleic acid; therefore, a list of desiderata for the sensitive detection of a specific target nucleic acid sequence is outlined below:
a) the process should preferably not require copying of the target sequence;
b) the process should preferably not involve multiple copying of long tracts of sequence;
c) the process should preferably be generally applicable to both DNA and RNA target sequences including specific sequences without discrete ends;
d) the signal should preferably result from the independent hybridisation of two different probes; or regions of probe, to a target sequence; and
e) the process should include an option for detection of hybridised probe without any additional processes.
A nucleic acid amplification process that fulfils the above desiderata is disclosed in WO 93/06240 (Cytocell Ltd). Two amplification processes are described, one thermal and one isothermal. Both the thermal and isothermal versions depend on the hybridisation of two nucleic acid probes of which regions are complementary to the target nucleic acid. Portions of said probes are capable of hybridising to the sequence of interest such that the probes are adjacent or substantially adjacent to one another, so as to enable complementary “arm” specific sequences of the first and second probes to become annealed to each other. Following annealing, chain extension of one of the probes is achieved by using part of the other probe as a template.
Amplification is achieved by one of two means; in the thermal cycling version thermal separation of the extended first probe is carried out to allow hybridisation of a further probe, substantially complementary to part of the newly synthesised sequence of the extended first probe. Extension of the further probe by use of an appropriate polymerase using the extended first probe as a template is achieved. Thermal separation of the extended first and further probe products allows these molecules to act as a template for the extension of further first probe molecules and the extended first probe can act as a template for the extension of other further probe molecules. In the isothermal version, primer extension of the first probe creates a functional RNA polymerase promoter that in the presence of a relevant RNA polymerase transcribes multiple copies of RNA. The resulting RNA is further amplified as a result of the interaction of complementary DNA oligonucleotides containing further RNA polymerase promoter sequences, whereupon annealing of the RNA on the DNA oligonucleotide and a subsequent extension reaction leads to a further round of RNA synthesis. This cyclical process generates large yields of RNA, detection of which can be achieved by a number of means. The present invention is related to these processes and aims to provide improvements thereon.
SUMMARY OF THE INVENTION
In preferred embodiments the present invention also fulfils all the aforementioned desiderata. This may be achieved through the hybridisation of two oligonucleotide probes that contain complementary target specific regions together with complementary arm regions, such that in the presence of the target sequence of interest the target and the two probes form a “three way junction”. Within the complementary arm region of one or both of the oligonucleotide probes is incorporated a destabilizing moiety that prevents the two oligonucleotide probes from asso
Assenberg Rene
Cardy Donald L. N.
Marsh Peter
Mock Graham A
Ray Trevor D
Cytocell Limited
Lu Frank
Whisenant Ethan C.
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