A method for assembling circular and linear dna molecules in an ordered manner
AssigneeUniversity of Marlyland, Baltimore County
InventorsCHARLES J. BIEBERICH, XIANG LI
Life of this patent
Estimating the term…
Abstract
The present invention relates to a method of assembling circular and linear DNA molecules, more specifically, the present invention provides for a homology-based, one-tube assembly method including a circular DNA vector and at least one restriction enzyme without prior linearization of such a circular DNA vector.
Claims20
A one pot method to prepare a circular or linear DNA molecule for use in preparing a nucleotide end-product, the method comprising:
- providing a reaction vessel, a combination of a circular DNA vector and an amplified linearized target DNA molecule;
- introducing into the reaction vessel at essentially the same time the circular DNA vector and the amplified linearized target DNA molecule and at least one restriction enzyme into the reaction vessel in an amount to linearize the circular DNA vector;
- adding to the reaction vessel a buffering solution, wherein the buffering solution comprises at least a DNA polymerase, a 5′-3′ exonuclease, a buffering agent and optionally a DNA ligase;
- incubating the circular DNA vector, the amplified linearized target DNA molecule and the buffering solution for a sufficient time and temperature for linearization of the circular DNA vector and joining the amplified linearized target DNA molecule and the linearized circular DNA vector for production of the circularized or linearized DNA molecule.
A one pot method to prepare a circular or linear DNA molecule, the method comprising:
- providing a reaction vessel, a circular plasmid and a PCR amplified product of a linearized target DNA molecule encoding a desired target protein;
- introducing into the reaction vessel at essentially the same time the circular plasmid and PCR amplified product of the linearized target DNA and at least two restriction enzymes into the reaction vessel in an amount to linearize the circular plasmid, wherein the restriction enzymes comprises a combination of BamHI and SalI;
- adding to the reaction vessel an incubating solution, wherein the incubation solution comprises components comprising at least a DNA polymerase, a 5′-3′ exonuclease, a buffering solution comprising at least a crowding agent such as a PEG molecule, dNTPs, potassium acetate, magnesium acetate, a tris acetate buffering agent, bovine serum albumin, and optionally a DNA ligase;
- incubating the components at a temperature and for a sufficient time for linearization of the circular plasmid and joining the PCR amplified product and the linearized circular plasmid for production of a circularized or linearized DNA molecule for subsequent expression in a host cell, wherein the incubation time and temperature is selected from the group 37° C. for 15 minutes +50° C. for about 15 minutes;
- 50° C. for 60 minutes;
- 37° C. for 15 minutes+50° C. for 45 minutes and 37° C. for 30 minutes +50° C. for 30 minutes.
A composition for a one pot synthesis of a circularized or linearized DNA molecule, the composition comprising;
- a circular DNA vector, a linear DNA molecule, at least one restriction enzyme. at least a DNA polymerase, a 5′-3′ exonuclease, an incubating solution comprising at least a crowding agent, dNTPs, and a tris buffering agent, and optionally a DNA ligase.
Dependencies are inferred from the claim text.