Assay involving looped nucleic acid

Details Assay involving looped nucleic acid Assay involving looped nucleic acid

1999-06-16

2003-05-20

Siew, Jeffrey (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C435S091210, C536S063000, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06566058

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method for detecting the presence of a nucleic acid sequence of interest, and to a kit of components for performing the assay method.
BACKGROUND OF THE INVENTION
A number of nucleic acid amplification processes are described in the prior art. One such well known process, polymerase chain reaction (PCR), is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202. The PCR process involves the use of nucleic acid primers which 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 in order to further amplify copies of the original nucleic acid sequence.
This method has some disadvantages 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 problem, 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 in WO 88/10315 (Siska Diagnostics) and European patent nos. 329,822 (Cangene) and 373,960 (Siska Diagnostics). These amplification processes are based on a reaction comprising alternate cycles of DNA and RNA synthesis. This alternating RNA/DNA synthesis is achieved principally through the annealing of oligonucleotides adjacent to a specific DNA sequence, whereby the annealed oligonucleotides comprise a transcriptional promoter and initiation site. The RNA copies of the specific sequence so produced, or alternatively an input sample comprising a specific RNA sequence, 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 329,882 and EP 373,960). 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 substantially a single temperature (i.e. isothermally). Additionally, a much greater level of amplification per cycle can be achieved than for PCR i.e. a doubling of DNA copies per cycle for PCR, compared with 10-100 RNA copies per cycle using T7 RNA polymerase. A disadvantage associated with the DNA:RNA cycling method described in EP 329,822 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 invariably 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 (RNA and/or DNA) 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 quantitating the original amount of specific nucleic acid.
The prior art methods described above have a number of disadvantages in the amplification of their target nucleic acid. It seems to the present inventors that a method for the sensitive detection of a specific target nucleic acid sequence should have the following characteristics:
a) the process should not necessarily require copying of the target sequence;
b) the process should not involve multiple copying of long tracts of sequence to minimise sequence errors;
c) the process should be generally applicable to both DNA and RNA target sequences, including specific sequences without discrete ends;
d) the signal should result from the two or more different hybridisation events so as to improve specificity;
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). This amplification process is centred around the use of two nucleic acid probes which contact the target nucleic acid, portions of said probes being capable of hybridising to the sequence of interest such that the probes are adjacent or substantially adjacent to one another, so as to enable other portions 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 of the extended probe is typically accomplished by: hybridisation of a further probe substantially complementary to part of the newly synthesised sequence of the extended first probe; extending the further probe by use of an appropriate polymerase using the extended first probe as a template; and separating the extended first and further probes, such that the extended further probe can act as a template for the extension of other first probe molecules, and the extended first probe can act as a template for the extension of other further probe molecules.
Other discolosures of interest include U.S. Pat. Nos. 5,451,503 and 5,424,413 (Gen-Probe, Inc.), which refer to the possibility of forming stem/loop structures upon hybridising a nucleic acid probe to a target sequence. The stem/loop may be formed in either the probe or the target sequence. The documents teach the detection of the duplex stem by various means. WO 97/19193 (Yale University, published May 29, 1997) describes a method of amplifying nucleic acid by means of “rolling circle replication”, which produces long concatameric copies of circular probe molecules. The preferred method involves the hybridisation of a linear probe molecule to a target sequence of interest, which brines together non-contiguous portions of the probe molecule, which are then joined in a ligation step to form closed circular nucleic acid molecules. A second probe is hybridised to the circular molecule to initiate rolling circle replication.
Detection of nucleic acid target sequences of interest may be useful clinically (e.g. detecting nucleic acid belonging to pathogens, thus aiding diagnosis of infectious disease, or detecting chromosomal abnormalities such as the “Philadelphia” chromosomal translocation associated with certain cancers), or in public health or environmental fields (e.g. detecting the presence of pathogens such as Salmonella spp in foodstuffs and the like, or detecting
E. coli,
an indicator of faecal contamination, in water supplies and the like).
It should also be mentioned that those skilled in the art are also acquainted with a substance known as PNA (or peptide nucleic acid). PNA comprises the conventional base compounds present in RNA or DNA. However, instead of the bases being covalently bound to a sugar/phosphate backbone, the bases are joined to a peptide backbone. PNA has many properties in common with RNA and DNA: for example, one can form chimeric duplex molecules by annealing a PNA strand to a strand of RNA or DNA. However, PNA also differs from conventional nucleic acid i

Affiliated with

Also associated with

Rating

Assay involving looped nucleic acid does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Assay involving looped nucleic acid, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Assay involving looped nucleic acid will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3014874