Method of nucleic acid sequence selection

Abstract

The present invention provides a method for the rapid isolation and recovery of a desired target DNA or RNA molecules from a mixture or library containing such molecules. The method involves the use of biotinylated probes and enzymatic repair-cleavage to eliminate undesired library members from a sample.

What is claimed is:  
     
         1 . A method for recovering a desired target nucleic acid molecule from a sample containing a mixture or library of single-stranded nucleic acid containing said molecule, wherein said method comprises the steps: 
 A. incubating said sample containing said nucleic acid mixture or library in the presence of a primer nucleic acid molecule complementary to a sequence of said desired target molecule; said incubation being under conditions sufficient to permit hybridization between said primer and said desired target molecule, and further sufficient to permit the template-dependent extension of said primer to thereby generate a double-stranded desired target molecule;    B. transforming single-stranded and double-stranded members of said mixture or library into a host cell, and    C. recovering said desired molecule from said cell.    
     
     
         2 . The method of  claim 1 , wherein prior to commencing step A, said method comprises the presteps: 
 (1) incubating an initial sample containing said nucleic acid mixture or library in the presence of a haptenylated nucleic acid probe molecule, said probe molecules having a sequence complimentary to a nucleotide sequence of said desired target molecule; said incubation being under conditions sufficient to permit said probe to hybridize to said desired target molecule and to thereby generate a hybridized molecule wherein said target molecule is bound to said probe;    (2) incubating said sample containing said nucleic acid mixture or library and biotinylated probe-target hybridized molecules of prestep (1) in the presence of a binding ligand of the hapten of said haptenylated probe, said binding ligand being conjugated to support; said incubation being sufficient to permit said probe molecules, and said probe-target hybridized molecule to become bound to said binding ligand of said support;    (3) recovering said probe-target hybridized molecules bound to said support from said nucleic acid mixture or library and any unbound biotinylated probe-target hybridized molecules of prestep (2); and    (4) incubating said recovered support containing said bound probe-target hybridized molecules under conditions sufficient to separate the strands of double-stranded molecules; said incubation thereby releasing said hybridized target molecule from said biotinylated probe, and generating a sample single-stranded desired target molecule for use in step (A).    
     
     
         3 . The method of  claim 1 , wherein said single-stranded nucleic acid molecule of said sample contains a nucleotide analog, and wherein after completing step A, but prior to commencing step B, said method additionally comprises the presteps: 
 (1′) incubating said generated double-stranded molecules in the presence of a nuclease capable of degrading nucleic acid containing nucleotide analog residues; and    (2′) incubating non-degraded nucleic acid with a primer under conditions sufficient to permit said primer to be extended in a template-dependent manner.    
     
     
         4 . The method of  claim 1 , wherein in step A, said template-dependent extension of said primer is conducted in the presence of a nuclease resistant nucleotide analog to thereby generate a double-stranded desired target molecule containing a residue of said nucleotide analog; and wherein prior to commencing said step B, said method additionally comprises the presteps: 
 (1″) incubating said generated double-stranded desired target molecule in the presence of a nuclease, wherein said nuclease is substantially unable to cleave a nucleic acid molecule containing said nucleotide analog residue, but is substantially capable of degrading both single-stranded nucleic acid molecules and double-stranded nucleic acid molecules that lack said nucleic acid analog residue; said incubation being under conditions sufficient to permit such degradation, and thereby substantially eliminating both single-stranded nucleic acid molecules and double-stranded nucleic acid molecules that lack said nucleic acid analog residue from said sample; and thereby forming a preparation having a substantial enrichment of said desired target molecule relative to said initial sample; and    (2″) recovering said desired molecule from said preparation of prestep (1″) to thereby form a library or mixture for said step B.    
     
     
         5 . The method of  claim 1 , wherein in step A said incubation is under conditions which minimize random hybridization.  
     
     
         6 . The method of  claim 2 , wherein in prestep (1) said desired incubation is under conditions which minimize random hybridization.  
     
     
         7 . The method of  claim 1 , wherein said desired target nucleic acid molecule is a DNA molecule.  
     
     
         8 . The method of  claim 7 , wherein said DNA molecule is a single-stranded DNA molecule.  
     
     
         9 . The method of  claim 1 , wherein said desired target nucleic acid molecule is an RNA molecule.  
     
     
         10 . The method of  claim 1 , wherein said desired target nucleic acid molecule is a single-stranded nucleic acid molecule.  
     
     
         11 . The method of  claim 1 , wherein said desired target molecule is a circular nucleic acid molecule.  
     
     
         12 . The method of  claim 11 , wherein said desired target molecule is a circular DNA molecule.  
     
     
         13 . The method of  claim 2 , wherein said hapten is biotin, and wherein said binding ligand of said hapten is avidin, streptavidin, or an antibody or antibody fragment that binds biotin.  
     
     
         14 . The method of  claim 13 , wherein said biotin-binding ligand is avidin.  
     
     
         15 . The method of  claim 13 , wherein said biotin-binding ligand is streptavidin.  
     
     
         16 . The method of  claim 2 , wherein said support of said prestep (2) is a paramagnetic bead.  
     
     
         17 . The method of  claim 16 , wherein said hybridized molecule bound to said paramagnetic bead is recovered by magnetic means.  
     
     
         18 . The method of  claim 2 , wherein in said primer molecule of step A is complementary to the same sequence of said desired target molecule as said probe molecule of substep (1).  
     
     
         19 . The method of  claim 2 , wherein in said primer molecule of step A is complementary to a sequence of said desired target molecule that differs from the sequence of said desired target molecule that is complementary to said probe molecule of substep (1).  
     
     
         20 . The method of  claim 3 , wherein said nucleic acid analog is deoxyuridine, and wherein said nuclease is UDG.  
     
     
         21 . The method of  claim 4 , wherein said nuclease does not cleave hemimethylated DNA.  
     
     
         22 . The method of  claim 21 , wherein said nucleic acid analog is 5-methylcytidine, and wherein said nuclease that does not cleave hemimethylated DNA is HhaI.  
     
     
         23 . The method of  claim 2 , wherein in prestep (1), said probe has a degenerate sequence.  
     
     
         24 . The method of  claim 4 , wherein in step A, said primer has a degenerate sequence.  
     
     
         25 . The method of  claim 1 , wherein said host cell is a bacterium.  
     
     
         26 . The method of  claim 1 , wherein said method additionally includes the step of amplifying said desired target molecule by an in vitro amplification reaction.  
     
     
         27 . The method of  claim 26 , wherein said in vitro amplification reaction is a polymerase chain reaction.