The present disclosure provides reactive quencher dyes that can be used in the detection and/or quantification of desirable target molecules, such as proteins, nucleic acids and various cellular organelles. These dyes are essentially non-fluorescent but are efficient quenchers of various fluorescent dyes. Also, provided are methods of using the dyes, bio-probes incorporating dyes and methods of using the bio-probes. The quencher dyes described herein are modified to provide beneficial properties.

There is provided a method for detecting a target RNA molecule in a sample comprising reverse transcription, amplification of the reverse transcription product, and detection of the amplification product, involving the use of (i) an RT oligonucleotide comprising a stem-loop portion containing one or more nucleotides modified or modifiable to block DNA polymerase extension and a target annealing portion that is complementary to a downstream portion of the target RNA, the target annealing portion located 3′ to the stem-loop portion, (ii) a first amplification primer that anneals to a downstream portion of a 3′ extended region of the reverse transcription product and (ii) a second amplification primer that anneals to an interface portion of a DNA strand complementary to the reverse transcription product, the interface portion comprising a region that is complementary to a 3′ portion of the RT oligonucleotide and a 5′ portion of the 3′ extended region in the reverse transcription product.

There is provided a method for detecting a target RNA molecule in a sample comprising reverse transcription, amplification of the reverse transcription product, and detection of the amplification product, involving the use of (i) an RT oligonucleotide comprising a stem-loop portion containing one or more nucleotides modified or modifiable to block DNA polymerase extension and a target annealing portion that is complementary to a downstream portion of the target RNA, the target annealing portion located 3′ to the stem-loop portion, (ii) a first amplification primer that anneals to a downstream portion of a 3′ extended region of the reverse transcription product and (ii) a second amplification primer that anneals to an interface portion of a DNA strand complementary to the reverse transcription product, the interface portion comprising a region that is complementary to a 3′ portion of the RT oligonucleotide and a 5′ portion of the 3′ extended region in the reverse transcription product.

Provided herein are methods of amplifying nucleic acids. In particular, methods are provided for amplifying circular RNA molecules. In certain embodiments, circular DNA molecules for amplification are generated from circular RNA molecules. Provided herein are methods for amplifying a nucleic acid. In certain embodiments, a method comprises priming a circular RNA template molecule with one or more DNA primers and extending the primers with a reverse transcriptase to generate a cDNA strand that is a copy of the circular RNA molecule. In certain embodiments, the cDNA strand generated is linear.

Provided herein are methods of amplifying nucleic acids. In particular, methods are provided for amplifying circular RNA molecules. In certain embodiments, circular DNA molecules for amplification are generated from circular RNA molecules. Provided herein are methods for amplifying a nucleic acid. In certain embodiments, a method comprises priming a circular RNA template molecule with one or more DNA primers and extending the primers with a reverse transcriptase to generate a cDNA strand that is a copy of the circular RNA molecule. In certain embodiments, the cDNA strand generated is linear.

Linear covalently closed vectors, and compositions and methods for making same.

Fluidic cartridge including a liquid container having a reservoir configured to hold a liquid. The liquid container includes an interior surface. The fluidic cartridge also includes a transfer tube that extends from the interior surface to a distal end. The distal end includes a fluidic port that is in flow communication with the reservoir through the transfer tube. The transfer tube has a piercing segment that includes the distal end. The fluidic cartridge also includes a movable seal that is engaged to the piercing segment of the transfer tube and configured to slide along the piercing segment from a closed position to a displaced position during a mating operation. The movable seal blocks flow of the liquid through the fluidic port when in the closed position. The piercing segment extends through the movable seal when in the displaced position.

An in vitro process for the production of closed linear deoxyribonucleic acid (DNA) comprises (a) contacting a DNA template comprising at least one protelomerase target sequence with at least one DNA polymerase in the presence of one or more primers under conditions promoting amplification of the template; and (b) contacting amplified DNA produced in (a) with at least one protelomerase under conditions promoting production of closed linear DNA. A kit provides components necessary in the process.

An in vitro process for the production of closed linear deoxyribonucleic acid (DNA) comprises (a) contacting a DNA template comprising at least one protelomerase target sequence with at least one DNA polymerase in the presence of one or more primers under conditions promoting amplification of the template; and (b) contacting amplified DNA produced in (a) with at least one protelomerase under conditions promoting production of closed linear DNA. A kit provides components necessary in the process.

The present invention discloses a nucleic acid extraction apparatus and a method using the apparatus for extracting nucleic acids and amplifying target nucleic acids. The nucleic acid extraction apparatus comprising: a nucleic acid extraction element, a waste storage chamber, and a reaction chamber, wherein the reaction chamber is selectively in communication with the nucleic acid extraction element and the waste storage chamber, and the apparatus realizes fluid exchange through the pressure between the waste storage chamber and the reaction chamber. In the present invention, by using an integrated control system, the conventional manual process of nucleic acid extraction and purification is integrated into a fully automatic closed process, making the operation more conveniently and quickly and improving the efficiency of experimental work.