Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes that comprise a ribonucleotide position that is susceptible to endoribonuclease (e.g., RNase H) cleavage in the presence of target nucleic acid molecules. Probes of the embodiments may also comprise non-natural nucleotide linked to a reporter and/or quenching moiety.

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes that comprise a ribonucleotide position that is susceptible to endoribonuclease (e.g., RNase H) cleavage in the presence of target nucleic acid molecules. Probes of the embodiments may also comprise non-natural nucleotide linked to a reporter and/or quenching moiety.

The present invention provides a method for easily and exponentially amplifying circular DNA, particularly long chain circular DNA, in a cell-free system. Specifically, the present invention provides a method for amplifying circular DNA in which circular DNA having a replication origin sequence (origin of chromosome (oriC)) is mixed with a reaction solution containing the following enzyme groups to form a reaction mixture, which is then reacted under an isothermal condition, the enzyme groups being:

(1) a first enzyme group that catalyzes replication of circular DNA;
(2) a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane; and
(3) a third enzyme group that catalyzes a separation of two sister circular DNAs.

The present invention provides a method for easily and exponentially amplifying circular DNA, particularly long chain circular DNA, in a cell-free system. Specifically, the present invention provides a method for amplifying circular DNA in which circular DNA having a replication origin sequence (origin of chromosome (oriC)) is mixed with a reaction solution containing the following enzyme groups to form a reaction mixture, which is then reacted under an isothermal condition, the enzyme groups being:

(1) a first enzyme group that catalyzes replication of circular DNA;
(2) a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane; and
(3) a third enzyme group that catalyzes a separation of two sister circular DNAs.

The embodiments disclosed herein utilize RNA targeting effectors to provide a robust CRISPR-based diagnostic for hemorrhagic fever virus applications. Embodiments disclosed herein can differentiate between hemorrhagic fever viruses that present with similar symptoms, as well as between strains of a hemorrhagic fever virus.

The embodiments disclosed herein utilize RNA targeting effectors to provide a robust CRISPR-based diagnostic for hemorrhagic fever virus applications. Embodiments disclosed herein can differentiate between hemorrhagic fever viruses that present with similar symptoms, as well as between strains of a hemorrhagic fever virus.

The invention can be used to provide a more efficient and less error-prone method of detecting variants in DNA, such as SNPs and indels. The invention also provides a method for performing inexpensive multiplex assays. The invention also provides methods for detection of DNA sequences altered after cleavage by a targetable endonuclease, such as the CRISPR Cas9 protein from the bacterium Streptococcus pyogenes.

The invention can be used to provide a more efficient and less error-prone method of detecting variants in DNA, such as SNPs and indels. The invention also provides a method for performing inexpensive multiplex assays. The invention also provides methods for detection of DNA sequences altered after cleavage by a targetable endonuclease, such as the CRISPR Cas9 protein from the bacterium Streptococcus pyogenes.

Kits and methods for enriching target nucleic acid sequences, such as nucleic acid molecules including the target nucleic acid sequence, and kits and methods for depleting target nucleic acid sequences, such as nucleic acid molecules including the target nucleic acid sequences. In an embodiment, the methods for enriching target nucleic acid sequences include selectively degrading single-stranded sample nucleic acid molecules, such as those that do not include the target nucleic acid sequences. In an embodiment, the methods for depleting target nucleic acid sequences include selectively degrading double-stranded sample nucleic acid molecules, such as those including the target nucleic acid sequence.

Kits and methods for enriching target nucleic acid sequences, such as nucleic acid molecules including the target nucleic acid sequence, and kits and methods for depleting target nucleic acid sequences, such as nucleic acid molecules including the target nucleic acid sequences. In an embodiment, the methods for enriching target nucleic acid sequences include selectively degrading single-stranded sample nucleic acid molecules, such as those that do not include the target nucleic acid sequences. In an embodiment, the methods for depleting target nucleic acid sequences include selectively degrading double-stranded sample nucleic acid molecules, such as those including the target nucleic acid sequence.