Method for directional cloning

Abstract

Described herein are techniques for directional cloning an insert DNA segments into a target vector. The techniques mix the target vector, the insert DNA segment, a restriction enzyme, and a DNA ligase to generate a recombinant DNA molecule.

What is claimed is: 
     
         1 . A method for directional cloning, the method comprising:
 providing a plurality of DNA segments for the directional cloning;   incubating a lentiviral vector, the plurality of DNA segments, restriction enzymes, and DNA ligases in a single container at a room temperature for a period less than 30 minutes to obtain the lentiviral vector comprising a recombinant DNA molecule comprising the plurality of DNA segments in a desired direction; and   mixing the lentiviral vector with a competent host cell.   
     
     
         2 . The method of  claim 1 , wherein a positive rate of the directional cloning is greater than 80%. 
     
     
         3 . The method of  claim 2 , wherein the number of the plurality of DNA segments is more than 2. 
     
     
         4 . The method of  claim 1 , wherein comprises amplifying DNA segments by polymerase chain reaction (PCR) to obtain the plurality of DNA segments, respectively, and the plurality of DNA segment are a plurality of modified DNA segment. 
     
     
         5 . The method of  claim 1 , wherein the restriction enzyme comprises a type II restriction enzyme. 
     
     
         6 . The method of  claim 5 , wherein the restriction enzymes comprise at least one of BsaI, BbsI, BsmBI, Alw26I, AarI, BsmAI, BsmFI, BspMI, BtgZI, SfaNI, or LguI. 
     
     
         7 . The method of  claim 1 , wherein the DNA ligases comprise at least one of T4 DNA ligase or  E. coli  DNA ligase. 
     
     
         8 . The method of  claim 1 , wherein an individual DNA segment of the plurality of DNA segments comprising adaptors, and the recombinant DNA molecule does not comprise adaptors. 
     
     
         9 . The method of  claim 1 , wherein the period is less than 15 minutes. 
     
     
         10 . The method of  claim 1 , wherein the period is about 10 minutes. 
     
     
         11 . A reaction mixture for use in directional cloning, the reaction mixture comprising a plurality of DNA segments, a lentiviral vector, a plurality of amplified DNA segments for the directional cloning, restriction enzymes, and DNA ligases, wherein the number of the plurality of DNA segments is more than 2, and the directional cloning is performed at a room temperature for a period less than 30 min. 
     
     
         12 . The reaction mixture of  claim 11 , wherein a positive rate of the directional cloning is greater than 80%. 
     
     
         13 . The reaction mixture of  claim 12 , wherein the number of the plurality of DNA segments is more than 2. 
     
     
         14 . The reaction mixture of  claim 11 , wherein comprises amplifying DNA segments by polymerase chain reaction (PCR) to obtain the plurality of DNA segments, respectively, and the plurality of DNA segment are a plurality of modified DNA segment. 
     
     
         15 . The reaction mixture of  claim 11 , wherein the restriction enzyme comprises a type II restriction enzyme. 
     
     
         16 . The reaction mixture of  claim 15 , wherein the restriction enzymes comprise at least one of BsaI, BbsI, BsmBI, Alw26I, AarI, BsmAI, BsmFI, BspMI, BtgZI, SfaNI, or LguI. 
     
     
         17 . The reaction mixture of  claim 11 , wherein the DNA ligases comprise at least one of T4 DNA ligase or  E. coli  DNA ligase. 
     
     
         18 . The reaction mixture of  claim 11 , wherein an individual DNA segment of the plurality of DNA segments comprising adaptors.