Amplification method for polynucleotide detection assays

#1. Field of the Invention

#Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the .sup.32 P labelling of DNA with T4 kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.

#Current methods for detecting specific nucleic acid sequences generally involve immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labelled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.

#FIG. 1 depicts a method for obtaining multiple copies of a primary polynucleotide sequence in accordance with the present invention.

#FIG. 2 depicts a method for effecting formation of a primary polynucleotide sequence in response to the presence of a polynucleotide analyte in accordance with the present invention.

#FIGS. 3-6 depict alternative methods for effecting formation of a primary polynucleotide sequence by the presence of a polynucleotide analyte.

#The present method allows the production of multiple copies of a primary polynucleotide sequence. The formation of the primary polynucleotide sequence can be initiated by the presence of a target polynucleotide sequence located at the 3'-terminus of a polynucleotide and is useful in detection of such a target sequence. The target polynucleotide sequence may be present in a polynucleotide analyte in a sample suspected of containing the analyte. The primary polynucleotide sequence may be the same as, but is preferably different from, the target polynucleotide sequence. In the production of multiple copies of a primary polynucleotide sequence the following components are provided: (i) the primary polynucleotide sequence, (ii) a single stranded pattern polynucleotide comprising two or more template sequences each containing one or more site specific cleavage sequences, (iii) nucleoside triphosphates, (iv) template-dependent polynucleotide polymerase, and (v) means for specifically cleaving cleavable polynucleotide sequences when an extension of the primary polynucleotide sequence is hybridized with the site specific cleavage sequences. The template sequence is complementary to and can hybridize with the primary polynucleotide sequence. The complex of the primary polynucleotide sequence with the single stranded pattern polynucleotide is incubated either simultaneously or wholly or partially sequentially with remaining components under conditions for either simultaneously or wholly or partially sequentially forming the extension of the primary polynucleotide sequence comprising one or more copies of the primary polynucleotide sequence connected through the cleavable polynucleotide sequences, cleaving the extension into fragments at the cleavable polynucleotide sequences, dissociating the complex of the template sequence and the cleaved primary polynucleotide sequence which is identical with the primary polynucleotide sequence, and reforming the complex of the primary polynucleotide sequence with the single stranded pattern polynucleotide which is usually in excess. The above steps are repeated until the desired number of copies are obtained.

#The target polynucleotide sequence can be part of a polynucleotide analyte that is to be detected. One aspect of the invention comprises a determination of such a polynucleotide analyte by causing the target polynucleotide sequence to initiate the above described method for producing multiple copies. In this method the target polynucleotide sequence in the polynucleotide analyte is caused to terminate in a 3' hydroxyl group when it is not already so terminated. This is usually effected by incubation with a restriction endonuclease. The target sequence is dissociated from complementary polynucleotides and caused to hybridize with the single stranded pattern polynucleotide which has a complementary binding polynucleotide sequence bound at the 3' end of the template sequence. The formation of multiple copies of the primary polynucleotide sequence is then carried out by an extension of the target sequence along the single stranded pattern polynucleotide and cleavage of the extension into fragments as described above for the hybrid of the primary polynucleotide sequence with the single stranded pattern polynucleotide. The cleaved fragments are detected, and their presence indicates the presence of the polynucleotide analyte in the sample. The hybridization, chain extension, and cleaving can be repeated until the single stranded polynucleotide or nucleoside triphosphates are exhausted or the template-dependent polynucleotide polymerase becomes inactive, but will preferably not be repeated after the ratio of the cleaved fragments induced by the target polynucleotide sequence to those non-specifically induced ceases to increase. The present method has application to the detection of both DNA and RNA sequences.

#One composition of the present invention is a single stranded pattern polynucleotide that comprises an oligomer consisting of three to one-hundred monomeric units. Each monomeric unit consists of an identical oligodeoxynucleotide template having from about eight to one hundred, preferably 10 to 100, bases and at least one restriction site. Preferably, the oligomer is substituted at the 3' end by a binding polynucleotide sequence of 15 or more bases that is complementary to a target polynucleotide sequence. The single stranded pattern polynucleotide is substituted at the 3' end with a chemical functionality that prevents chain extension with a template dependent polynucleotide polymerase, or is cyclic.