Methods for preparing microRNAs from microvesicles isolated from a biological sample from a subject, and preparation of DNA from microvesicle microRNA preparations.

Methods and compositions are provided herein for preparing high-throughput cDNA sequencing libraries.

Methods and compositions are provided herein for preparing high-throughput cDNA sequencing libraries.

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 present invention shows that the measurement of the inhibitory activity of a subject's RBCs-RI (Red Blood Cells cytosolic ribonucleic inhibitors) on the blood/serum RNases is a novel way of differentiating healthy from septic blood and represents a valid biomarker with great clinical potential. In particular, the effect of lysing the erythrocytes of a blood biological sample and the subsequent release of the cytosolic ribonuclease inhibitors (RI), in particular the Blood ERythrocyte-derived RNase Inhibitors (BERRI), provides for a significant decrease in RNase activity in control or uninfected samples, while little effect is observed in septic samples, showing that the reduction of nuclease activity in the control or uninfected samples is due to the specific inhibition, by the BERRI, of the blood/serum RNases, in particular of the RNase A type endonuclease.

The present invention shows that the measurement of the inhibitory activity of a subject's RBCs-RI (Red Blood Cells cytosolic ribonucleic inhibitors) on the blood/serum RNases is a novel way of differentiating healthy from septic blood and represents a valid biomarker with great clinical potential. In particular, the effect of lysing the erythrocytes of a blood biological sample and the subsequent release of the cytosolic ribonuclease inhibitors (RI), in particular the Blood ERythrocyte-derived RNase Inhibitors (BERRI), provides for a significant decrease in RNase activity in control or uninfected samples, while little effect is observed in septic samples, showing that the reduction of nuclease activity in the control or uninfected samples is due to the specific inhibition, by the BERRI, of the blood/serum RNases, in particular of the RNase A type endonuclease.

Methods for enhancing specificity of an analyte binding moiety or probe oligonucleotide to an analyte are provided herein. For example, methods provided herein include blocking a capture binding domain, thereby preventing hybridization to the capture domain of the capture probe affixed to a substrate. Further methods include releasing the block from the capture binding domain, thereby allowing the capture binding domain to specifically bind to the capture domain of the capture probe on the substrate.

A fluorescent cross-linked RNase H mutant conjugate, a miRNA combination and an application thereof. The fluorescent cross-linked RNase H mutant conjugate (i) is as represented by RNase Hv-(Lx-SH-F)n or (ii) comprises an RNase Hv-Lx-ligand and receptor-F, wherein the ligand can bind to the receptor, RNase Hv is an RNase H mutant, which can bind to RNA or RNA-DNA hybrid strands, but cannot cleave RNA; L is a linker, and x is 1-10; SH is an amino acid containing a sulfhydryl group; F is a luminescent functional group, and n is 1-7. The fluorescent cross-linked RNase H mutant conjugate can directly recognize DNA/RNA hybrid strands and can be converted to generate detectable signals without PCR amplification, and can be applied to sensitively and quickly detect RNA.

A fluorescent cross-linked RNase H mutant conjugate, a miRNA combination and an application thereof. The fluorescent cross-linked RNase H mutant conjugate (i) is as represented by RNase Hv-(Lx-SH-F)n or (ii) comprises an RNase Hv-Lx-ligand and receptor-F, wherein the ligand can bind to the receptor, RNase Hv is an RNase H mutant, which can bind to RNA or RNA-DNA hybrid strands, but cannot cleave RNA; L is a linker, and x is 1-10; SH is an amino acid containing a sulfhydryl group; F is a luminescent functional group, and n is 1-7. The fluorescent cross-linked RNase H mutant conjugate can directly recognize DNA/RNA hybrid strands and can be converted to generate detectable signals without PCR amplification, and can be applied to sensitively and quickly detect RNA.