The present invention relates to the detection of a target nucleic acid sequence by a POCH (PO Cleavage and Hybridization) assay on a solid substrate. The present invention detects the target nucleic acid sequence by use of in which the PO (Probing Oligonucleotide) hybridized with the target nucleic acid sequence is cleaved and the cleavage of the PO is detected by hybridization with the CO (Capturing Oligonucleotide). In the present invention, an uncleaved PO is hybridized with the CO immobilized onto the solid substrate. The designs of the PO and the CO are convenient and the optimization of reaction conditions is routinely easy in the present invention. Where the detection of signal on the solid substrate is continuously performed along with repetition of cleavage of the POs in the present invention, the number of the POs cleaved is increased upon the repetition number of the cleavage reaction and the signal is changed in parallel with the number of the POs cleaved. Then, the target nucleic acid sequence can be detected in a real-time manner. In contrast, the change of the signal is not observed in the absence of the target nucleic acid sequence.

A method and reagent for constructing a nucleic acid double-joint single-strand cyclical library. The method comprises: breaking a nucleic acid into nucleic acid fragments; connecting a first linker sequence; producing by amplification a first product provided with the first linker sequence at either end, where a U nucleobase is provided on a primer sequence; using USER enzyme to cleave the first product and cyclizing to produce a gap; or, a nicking enzyme recognition sequence is also provided on the primer sequence, using the USER enzyme to cleave the first product, cyclizing and using a nicking enzyme for nicking to produce a nick; performing a restrictive nick/gap translation reaction from the nick or the gap; removing by digestion any portion that did not undergo the restrictive nick/gap translation reaction; connecting a second linker sequence; producing by amplification a second product provided with the second linker sequence at either end; denaturing the second product, and using a mediated sequence for cyclization of a single-strand nucleic acid molecule. The method allows an increase in the length of library insert fragments and obviates the need for gel extraction; the single-strand nucleic acid molecule can be cyclized directly when denatured with heat.

A method and reagent for constructing a nucleic acid double-joint single-strand cyclical library. The method comprises: breaking a nucleic acid into nucleic acid fragments; connecting a first linker sequence; producing by amplification a first product provided with the first linker sequence at either end, where a U nucleobase is provided on a primer sequence; using USER enzyme to cleave the first product and cyclizing to produce a gap; or, a nicking enzyme recognition sequence is also provided on the primer sequence, using the USER enzyme to cleave the first product, cyclizing and using a nicking enzyme for nicking to produce a nick; performing a restrictive nick/gap translation reaction from the nick or the gap; removing by digestion any portion that did not undergo the restrictive nick/gap translation reaction; connecting a second linker sequence; producing by amplification a second product provided with the second linker sequence at either end; denaturing the second product, and using a mediated sequence for cyclization of a single-strand nucleic acid molecule. The method allows an increase in the length of library insert fragments and obviates the need for gel extraction; the single-strand nucleic acid molecule can be cyclized directly when denatured with heat.

Methods of producing single-stranded deoxyribonucleic acids (ssDNAs) are provided. Aspects of the methods include generating a double stranded deoxyribonucleic acid (dsDNA) and then selectively degrading one strand of the dsDNA to produce a ssDNA. ssDNAs produced using methods of the invention find use in a variety of applications, including genomic modification applications. Also provided are compositions, e.g., kits, that find use in practicing various embodiments of the invention.

Methods of producing single-stranded deoxyribonucleic acids (ssDNAs) are provided. Aspects of the methods include generating a double stranded deoxyribonucleic acid (dsDNA) and then selectively degrading one strand of the dsDNA to produce a ssDNA. ssDNAs produced using methods of the invention find use in a variety of applications, including genomic modification applications. Also provided are compositions, e.g., kits, that find use in practicing various embodiments of the invention.

The invention is a novel method of separately sequencing each strand of a nucleic acid involving the use of an adaptor comprising a strand cleavage site or a strand synthesis termination site. The adaptor may also be self-priming at the strand cleavage site.

The invention is a novel method of separately sequencing each strand of a nucleic acid involving the use of an adaptor comprising a strand cleavage site or a strand synthesis termination site. The adaptor may also be self-priming at the strand cleavage site.

A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterized by the steps of (1) generating a stream of single nucleotide bases from the analyte by pyrophosphorolysis; (2) producing captured molecules by reacting each single nucleotide base with a capture system labelled with detectable elements in an undetectable state; (3) releasing the detectable elements from each captured molecule in a detectable state and (4) detecting the detectable elements so released and determining the sequence of nucleotide bases therefrom. The method can be used advantageously in sequencers involving the use of microdroplets.

A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterized by the steps of (1) generating a stream of single nucleotide bases from the analyte by pyrophosphorolysis; (2) producing captured molecules by reacting each single nucleotide base with a capture system labelled with detectable elements in an undetectable state; (3) releasing the detectable elements from each captured molecule in a detectable state and (4) detecting the detectable elements so released and determining the sequence of nucleotide bases therefrom. The method can be used advantageously in sequencers involving the use of microdroplets.

The present invention provides for novel methods and compositions for nucleic acid sequence detection. Unique, identifying cleavage fragments from probes, bound to target nucleic acids, are produced during PCR by the 5′-nuclease activity of the polymerase. The identity of the targets can be determined by identifying the unique cleavage fragments.