Amplification-based cloning method

Abstract

The invention provides a method for isolating a nucleic acid molecule of interest from a nucleic acid library, which involves generating multiple copies of a nucleic acid molecule of interest present in the library and an enrichment step to remove template nucleic acid molecules. The method of invention has greater efficiency and possesses superior features compared to conventional cloning methods, and is capable of use for identifying and isolating known and novel nucleic acid molecules.

What is claimed is:  
     
         1 . A method of isolating a nucleic acid molecule of interest from a mixture of nucleic acid molecules, comprising: 
 (i) providing a recombinant nucleic acid library comprising a heterogeneous population of circular template nucleic acid molecules modified such that they are selectively digestible by an enzyme;    (ii) contacting the library with a first primer and a second primer, said primers capable of annealing to complementary strands of a nucleic acid molecule of interest present in the library to produce an annealed mixture, and wherein 
 the two primers extend in opposite directions relative to each other during primer extension; and  
 the 5′ ends of the two primers are adjacent to each other;  
   (iii) subjecting the annealed mixture to conditions under which primer extension occurs, thereby producing a reaction mixture containing linear primer extension products;    (iv) digesting the mixture containing linear primer extension products with the enzyme of (i), wherein said enzyme selectively digests template nucleic acid molecules but not the primer extension products, resulting in a population enriched for the nucleic acid molecule of interest.    
     
     
         2 . The method of  claim 1 , wherein the two primers are phosphorylated on their 5′ ends.  
     
     
         3 . The method of  claim 2 , further comprising ligating the linear primer extension products prior to the digesting step to produce circular primer extension products.  
     
     
         4 . The method of  claim 2 , further comprising, after the digesting step, isolating the primer extension products and ligating the isolated products.  
     
     
         5 . The method of  claim 4 , wherein the primer extension products are isolated by gel purification.  
     
     
         6 . The method of  claim 1 , wherein the enzyme is a restriction endonuclease.  
     
     
         7 . The method of  claim 6 , wherein the template circular nucleic acid molecules of (i) are methylated and the enzyme is a restriction endonuclease.  
     
     
         8 . The method of  claim 1 , wherein the library comprises nucleic acid molecules obtained from two or more tissue sources.  
     
     
         9 . The method of  claim 1 , wherein the nucleic acid library is a double-stranded DNA library.  
     
     
         10 . The method of  claim 1 , wherein the nucleic acid library is a human cDNA library.  
     
     
         11 . The method of  claim 1 , wherein the recombinant DNA library is a human genomic DNA library.  
     
     
         12 . The method of  claim 1 , wherein the nucleic acid molecule of interest is represented in the library at a frequency of equal to or less than one in 5×10 5  clones.  
     
     
         13 . The method of  claim 1 , wherein the nucleic acid molecule of interest is represented in the library at a frequency of equal to or less than one in 1×10 6  clones.  
     
     
         14 . The method of  claim 3 , further comprising transforming the circular primer extension products into a suitable host cell after the digesting step, to generate clones.  
     
     
         15 . The method of  claim 14 , wherein the host cell is a competent bacterial host cell.  
     
     
         16 . The method of  claim 14 , further comprising screening the clones to identify the clone containing the nucleic acid molecule of interest.  
     
     
         17 . The method of  claim 14 , further comprising sequencing the nucleic acid isolated from the clones.  
     
     
         18 . The method of  claim 8 , wherein the library is provided as a microarray and two or more nucleic acid molecules of interest are isolated simultaneously.  
     
     
         19 . The method of  claim 1 , wherein multiple DNA libraries are provided in a single mix.  
     
     
         20 . The method of  claim 19 , wherein greater than 50 cDNA libraries are in the single mix.