Array based methods for synthesizing nucleic acid mixtures

Abstract

Methods for generating mixtures of nucleic acids, e.g., oligonucleotide primers, are provided. In the subject methods, an array is employed as template to generate mixtures of nucleic acids via a template driven primer extension reaction. In preferred embodiments, each probe on the array employed in the subject methods comprises a constant domain and a variable domain, where the constant domain is further characterized by having at least a recognition domain. Also provided are the arrays employed in the subject methods and kits for practicing the subject methods. The subject methods find use in a variety of applications, including the generation of target nucleic acids from an mRNA sample for use in hybridization assays, e.g., differential gene expression analyses.

1 - 21 . (canceled)  
     
     
         22 . A method for producing a mixture of nucleic acids, said method comprising: 
 (a) providing an array of distinct single-stranded probe nucleic acids of differing sequence immobilized on a surface of a planar substrate where each distinct probe present on said array comprises a constant domain and a complement variable domain; wherein said complement variable domain is at the 5′ end of said each distinct probe;    (b) hybridizing nucleic acids complementary to said constant domain with said array of single-stranded probe nucleic acids to produce a template array of overhang comprising duplex nucleic acids, wherein each overhang comprising duplex nucleic acid of said array comprises a double-stranded constant region and a single-stranded variable region overhang;    (c) subjecting said template array of overhang comprising duplex nucleic acids to a cyclic reaction or an in vitro transcription protocol to produce a mixture of single stranded nucleic acids of differing sequence; and    (d) separating said mixture of nucleic acids from said template array.    
     
     
         23 . The method according to  claim 22 , wherein said mixture of nucleic acids is a mixture of deoxyribo-oligonucleotides.  
     
     
         24 . The method according to  claim 22 , wherein said step (c) comprises a cyclic reaction.  
     
     
         25 . The method according to  claim 24 , wherein said cyclic reaction comprises a protocol selected from the group consisting of: linear PCR and strand displacement amplification.  
     
     
         26 . The method according to  claim 22 , wherein said constant domain comprises at least one domain selected from the group consisting of: a linker domain; a functional domain and a recognition domain.  
     
     
         27 . The method according to  claim 22 , wherein said step (c) comprises an in vitro transcription protocol.  
     
     
         28 . The method according to  claim 27 , wherein said constant domain comprises at least one domain selected from the group consisting of: a linker domain; a functional domain and a recognition domain.  
     
     
         29 . The method according to  claim 28 , wherein said functional domain is an RNA polymerase promoter domain.  
     
     
         30 . The method according to  claim 22 , wherein said array is described by the formula:  
         surface-L-R—F-cV-5′ 
       wherein: 
 L is an optional linking domain;  
 R is a recognition domain;  
 F is a functional domain; and  
 cV is said complement domain.  
 
     
     
         31 . The method according to  claim 30 , wherein said hybridizing step (b) comprises contacting said array with a population of nucleic acids of the formula:  
         5′-cR-cF-3′ 
       wherein: 
 cR is the complement of R; and  
 cF is the complement of F.  
 
     
     
         32 . The method according to  claim 31 , wherein said template array of overhang comprising duplex nucleic acids is described by the formula:  
       
         
           
           
               
               
           
         
       
     
     
         33 . The method according to  claim 32 , wherein each distinct constituent member of said mixture produced by said method comprises a different variable domain V.  
     
     
         34 . The method according to  claim 30 , wherein said recognition domain is recognized by a restriction endonuclease.  
     
     
         35 . The method according to  claim 22 , wherein said array comprises at least about 50 different single-stranded probe nucleic acids of differing sequence.  
     
     
         36 . The method according to  claim 35 , wherein said mixture of nucleic acids produced by said method comprises at least about 50 nucleic acids of differing sequence.  
     
     
         37 . The method according to  claim 36 , wherein each constituent member of said mixture ranges in length from about 20 to 60 nt.  
     
     
         38 . A method according to  claim 22 , wherein said method further comprises employing said mixture of nucleic acids as primers in a target generation step in which target nucleic acids are produced from an mRNA sample to produce a population of target nucleic acids.  
     
     
         39 . The method according to  claim 38 , wherein said target generation step (b) comprises a template driven primer extension reaction.  
     
     
         40 . The method according to  claim 38 , wherein said target generation step (b) produces labeled target nucleic acids.  
     
     
         41 . The method according to  claim 38 , wherein said method further comprises contacting said set of target nucleic acids with an array of probe nucleic acids under hybridization conditions and detecting the presence of target nucleic acids hybridized to probe nucleic acids of said array.  
     
     
         42 . The method according to  claim 41 , wherein said target nucleic acids are labeled.  
     
     
         43 . The method according to  claim 41 , wherein said method further comprises washing unbound target away from the surface of said array.