Abstract

Provided herein is a method for efficiently capping RNA in vitro. In some embodiments the capping reaction may be done at high temperature using Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reactions may comprise a capping enzyme from a large virus of amoeba, e.g., Faustovirus, mimivirus or moumou virus, or a variant thereof. Compositions and kits for practicing the method are also provided.

IPC Classes  ?

• C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Abstract

Methods and compositions for capping RNA in an in vitro transcription (IVT) mixture are provided that include a thermostable RNA polymerase variant and a cap analog such that when a DNA template is added to the mixture, and the mixture is then incubated under conditions for in vitro transcription, capped RNA is produced. The methods describe high capping efficiencies at elevated temperatures as well as at standard temperatures for IVT. The RNA polymerase used is a thermostable variant of the T7 RNA polymerase, named polymerase M20, which comprises substitutions at positions 388 and 567, in particular D388E and V567P.

IPC Classes  ?

• C12N 9/12 - Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
• C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
• C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

Methods and compositions are provided for repairing a polynucleotide so that it can be copied with improved fidelity and/or yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a ligase and a cofactor selected from NAD+ or ATP and incubating the polynucleotide with the reaction mixture in the absence of Endonuclease VI. The reaction mixture may further contain an AP endonuclease and a polymerase. If used, these enzymes may be selected according to their ability to withstand high temperatures. For example, the reaction mixture may be used prior to a polynucleotide synthesis reaction in which case enzymes that are not thermophilic may be used. The repair reaction is not time sensitive with respect to seconds, minutes or hours of incubation in the enzyme mixture in as much as the repair is effected rapidly and prolonged incubation is not generally adverse.