Method for isolating and recovering target DNA or RNA molecules having a desired nucleotide sequence

Abstract

The invention generally concerns the use of amino acid denaturants for denaturing or separating double stranded nucleic acid molecules. More specifically, 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 haptenylated probes and amino add denaturants to select the desired molecules and eliminate the undesired library members from a sample. The invention also provides a method in which larger or full-length nucleic acid molecules can be isolated from the subpopulation of desired molecules.

1 - 40 . (canceled)  
     
     
         41 . A method of enriching one or more desired target nucleic acid molecules from a population of nucleic acid molecules comprising: 
 a) denaturing or separating double-stranded nucleic acid molecules within said population of nucleic acid molecules by contacting one or more double-stranded nucleic acid molecules with a denaturant selected from the group consisting of one or more amino acid denaturants, imidazole, and one or more amino acid denaturants plus imidazole, thereby forming single-stranded nucleic acid molecules;    b) contacting said single-stranded nucleic acid molecules with one or more primers complementary to one or more sequences of the desired target nucleic acid molecules; and    c) amplifying said desired target nucleic acid molecules;    wherein said desired target nucleic acid molecules are enriched.    
     
     
         42 . The method of  claim 41 , further comprising incubating said single-stranded nucleic acid molecules and said one or more primers with one or more nucleotides and a polypeptide having polymerase activity under conditions to generate one or more synthesized nucleic acid molecules complementary to said desired target molecules, thereby forming double-stranded nucleic acid molecules.  
     
     
         43 . The method of  claim 42 , wherein said nucleotides confer nuclease resistance to said one or more synthesized nucleic acid molecules.  
     
     
         44 . The method of  claim 43 , wherein said nucleotides are nucleotide analogs.  
     
     
         45 . The method of  claim 44 , wherein said nucleotide analogs are methylated nucleotides.  
     
     
         46 . The method of  claim 45 , wherein said methylated nucleotides are 5-methyldeoxycytosine.  
     
     
         47 . The method of  claim 46 , further comprising digesting one strand of said double-stranded nucleic acid molecules with one or more nucleases, thereby forming digested molecules.  
     
     
         48 . The method of  claim 47 , further comprising transforming said digested molecules into one or more host cells.  
     
     
         49 . The method of  claim 42 , further comprising transforming said double-stranded molecules into one or more host cells.  
     
     
         50 . The method of  claim 41 , wherein said amino acid denaturants are selected from the group consisting of one or more amino acids, polyamino acids, and combinations thereof; wherein said amino acid denaturants denature or separate double-stranded nucleic acid molecules.  
     
     
         51 . The method of  claim 50 , wherein said amino acid denaturants are natural or unnatural amino acids.  
     
     
         52 . The method of  claim 51 , wherein said amino acid denaturants are selected from the group consisting of glycine, D-alanine, L-alanine, DL-alanine, arginine, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.  
     
     
         53 . The method of  claim 52 , wherein said denaturant is glycine.  
     
     
         54 . The method of  claim 51 , wherein said amino acid denaturants comprise two or more amino acids.  
     
     
         55 . The method of  claim 41 , wherein the concentration of said denaturant ranges from about 1 mM to about 500 mM.  
     
     
         56 . The method of  claim 55 , wherein said concentration ranges from about 5 mM to about 50 mM.  
     
     
         57 . The method of  claim 56 , wherein said concentration is about 10 mM.  
     
     
         58 . The method of  claim 41 , wherein said one or more primers are degenerate primers.  
     
     
         59 . The method of  claim 58 , wherein said degenerate primers comprise one or more universal nucleotides.  
     
     
         60 . The method of  claim 59 , wherein said degenerate primers comprise one or more nucleotides selected from the group consisting of dP and dK.  
     
     
         61 . The method of  claim 41 , further comprising selecting or enriching for desired target nucleic acid molecules having larger or longer segments from a population of desired target nucleic acid molecules.  
     
     
         62 . The method of  claim 61 , wherein said enriching comprises separating the desired nucleic acid molecules according to size.  
     
     
         63 . The method of  claim 62 , wherein said method comprises amplifying the desired nucleic acid molecules prior to size separation.  
     
     
         64 . The method of  claim 41 , further comprising degradation of one strand of said double-stranded nucleic acid molecules.  
     
     
         65 . The method of  claim 64 , wherein said degradation comprises the use of Gene II protein and Exonuclease III.  
     
     
         66 . The method of  claim 41 , wherein said one or more primers comprise a Kozac sequence.  
     
     
         67 . The method of  claim 66 , wherein said one or more primers are degenerate primers.  
     
     
         68 . The method of  claim 41 , further comprising separating said one or more double-stranded nucleotide molecules according to size.  
     
     
         69 . The method of  claim 41  with the proviso that said denaturant is not selected from the group consisting of asparagine and β-alanine.  
     
     
         70 . The method of  claim 41 , wherein said population of nucleic acid molecules comprises circular DNA, linear DNA, or a cDNA library.