De novo synthesized plasmid, methods of making and use thereof

Abstract

The invention relates to a de novo synthesized plasmid. The plasmid comprises relevant sequences for plasmid replication and plasmid selection. The methods of making and use of the plasmid are disclosed. The plasmid can be used to make other plasmids. These plasmids and their host cells can be used for biomedical applications.

1 . A method of preparing a novel nucleic acid molecule capable of autonomous replication in a host cell comprising:
 (a) preparing a replication origin DNA fragment incapable of autonomous replication in a host cell by itself;   (b) preparing a selection marker DNA fragment;   (c) combining the DNA fragments prepared from steps (a) and (b) to form a novel nucleic acid molecule;   (d) introducing the nucleic acid molecule made from step (c) into a host cell; and   (e) selecting the nucleic acid molecule with appropriate replication origin and selection marker from the amplified host cells.   
     
     
         2 . The method according to  claim 1 , wherein the DNA fragment of step (a) or (b) is prepared from polymerase chain reaction. 
     
     
         3 . The method according to  claim 1 , wherein the DNA fragment of step (a) or (b) is prepared from restriction digestion. 
     
     
         4 . The method according to  claim 1 , wherein the DNA fragment of step (a) or (b) is prepared from synthetic nucleic acid. 
     
     
         5 . A method of combinatorially preparing a novel nucleic acid molecule combined from at least two sets of DNA fragments comprising:
 (a) preparing a set of at least two DNA fragments containing replication origins;   (b) preparing a set of at least two DNA fragments containing selection markers;   (c) combinatorially combining one DNA fragment prepared from step (a) and one DNA fragment prepared from step (b) to form a novel nucleic acid molecule;   (d) introducing the nucleic acid molecule made from step (c) into a host cell; and   (e) selecting the nucleic acid molecule with appropriate replication origin and selection marker from the amplified host cells.   
     
     
         6 . The method according to  claim 5 , wherein the DNA fragments of step (a) or (b) are prepared from polymerase chain reaction. 
     
     
         7 . The method according to  claim 5 , wherein the DNA fragments of step (a) or (b) are prepared from restriction digestion. 
     
     
         8 . The method according to  claim 5 , wherein the DNA fragments of step (a) or (b) are prepared from synthetic nucleic acid. 
     
     
         9 . A method of combinatorially preparing a novel nucleic acid molecule combined from at least two sets of DNA fragments comprising:
 (a) preparing at least two sets of DNA fragments;   (b) one set of the DNA fragments prepared from step (a) contains replication origin;   (c) one set of the DNA fragments prepared from step (a) contains at least two different DNA fragments;   (d) combinatorially combining two DNA fragments prepared from each set of step (a) to form a novel nucleic acid molecule;   (e) introducing the nucleic acid molecule made from step (d) into a host cell; and   (f) selecting the plasmid with appropriate replication origin and selection marker from the amplified host cells.   
     
     
         10 . The method according to  claim 9 , wherein the DNA fragments of step (a) are prepared from polymerase chain reaction. 
     
     
         11 . The method according to  claim 9 , wherein the DNA fragments of step (a) are prepared from restriction digestion. 
     
     
         12 . The method according to  claim 9 , wherein the DNA fragments of step (a) are prepared from synthetic nucleic acids.