The present invention relates to improved processes for production of closed linear deoxyribonucleic acid (DNA), in particular cell-free enzymatic production of closed linear DNA molecules, preferably using a closed linear DNA as a template for DNA synthesis. The invention further relates to a novel closed linear DNA species, suitable for use as a template in the improved processes for production of closed linear DNA. Further, the invention pertains to the intermediate products of the processes, since this enables the production of larger quantities of closed linear DNA from the template than with methods known in the art.

The present invention relates to improved processes for production of closed linear deoxyribonucleic acid (DNA), in particular cell-free enzymatic production of closed linear DNA molecules, preferably using a closed linear DNA as a template for DNA synthesis. The invention further relates to a novel closed linear DNA species, suitable for use as a template in the improved processes for production of closed linear DNA. Further, the invention pertains to the intermediate products of the processes, since this enables the production of larger quantities of closed linear DNA from the template than with methods known in the art.

The current invention pertains to adapters comprising a protelomerase recognition sequence, preferably a TeIN protelomerase recognition sequence. The adapters of the invention can be used for the preparation of a nucleic acid molecule library. The invention also relates to a method for producing a nucleic acid molecule library using one or more adapters comprising a protelomerase recognition sequence. The adapters may be contacted with a protelomerase to cleave and close the ends of the adapters. Said closed adapters are e.g. protected against exonuclease treatment. The method of the invention further concerns an amplification method and a sequencing method using adapters having a protelomerase recognition sequence.

The current invention pertains to adapters comprising a protelomerase recognition sequence, preferably a TeIN protelomerase recognition sequence. The adapters of the invention can be used for the preparation of a nucleic acid molecule library. The invention also relates to a method for producing a nucleic acid molecule library using one or more adapters comprising a protelomerase recognition sequence. The adapters may be contacted with a protelomerase to cleave and close the ends of the adapters. Said closed adapters are e.g. protected against exonuclease treatment. The method of the invention further concerns an amplification method and a sequencing method using adapters having a protelomerase recognition sequence.

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.

Provided herein are methods for generating circular nucleic acid molecules and circular nucleic acid libraries. The methods can be used to generate clonal populations of target nucleic acid molecules for downstream applications such as sequencing.

Provided herein are methods for generating circular nucleic acid molecules and circular nucleic acid libraries. The methods can be used to generate clonal populations of target nucleic acid molecules for downstream applications such as sequencing.

The present invention provides a method for determining whether or not an aqueous solution contains two or more cancer cells. The present method is characterized by the following three matters. First, the PCR solution contains the TS primer at a concentration of not less than 0.1 μM and not more than 1 μM in the present invention. Second, the PCR solution contains an ACX reverse primer. Third, the PCR solution contains the ACX reverse primer at a concentration of not less than 0.02 μM and not more than 0.06 μM in the present invention. In the present method, it is determined that an aqueous solution contains cancer cells even if the aqueous solution contains only two cancer cells.

The present invention provides a method for determining whether or not an aqueous solution contains two or more cancer cells. The present method is characterized by the following three matters. First, the PCR solution contains the TS primer at a concentration of not less than 0.1 μM and not more than 1 μM in the present invention. Second, the PCR solution contains an ACX reverse primer. Third, the PCR solution contains the ACX reverse primer at a concentration of not less than 0.02 μM and not more than 0.06 μM in the present invention. In the present method, it is determined that an aqueous solution contains cancer cells even if the aqueous solution contains only two cancer cells.