Methods of Cloning and Producing Fragment Chains with Readable Information Content

Abstract

The present invention provides a method of attaching a fragment of a first nucleic acid molecule to a second nucleic acid molecule using adapters to mediate the binding particularly in methods of cloning, methods of producing fragment chains with a readily readable information content, particularly comprising fragments corresponding to code, such as alphanumeric code, the nucleic acid molecules thus produced and kits for performing such methods.

1 . A method of synthesizing a double stranded nucleic acid molecule comprising at least the steps of: 
 1) generating n double stranded nucleic acid fragments, wherein at least n−2 fragments have single stranded regions at both termini and 2 fragments have single stranded regions at least one terminus, wherein (n−1) single stranded regions are complementary to (n−1) other single stranded regions, thereby producing (n−1) complementary pairs,    2) contacting said n double stranded nucleic acid fragments, simultaneously or consecutively, to effect binding of said complementary pairs of single stranded regions, and    3) optionally ligating said complementary pairs simultaneously or consecutively to produce a nucleic acid molecule consisting of n fragments,    wherein said fragment comprises a region representing a unit of information corresponding to one or more code elements and said code is alphanumeric.    
     
     
         2 . A method of synthesizing a double stranded nucleic acid molecule comprising at least the steps of: 
 1) generating n double stranded nucleic acid fragments, wherein at least n−2 fragments have single stranded regions at both termini and 2 fragments have single stranded regions at least one terminus, wherein (n−1) single stranded regions are complementary to (n−1) other single stranded regions, thereby producing (n−1) complementary pairs,    2) contacting said n double stranded nucleic acid fragments, simultaneously or consecutively, to effect binding of said complementary pairs of single stranded regions, and    3) optionally ligating said complementary pairs simultaneously or consecutively to produce a nucleic acid molecule consisting of n fragments,    wherein said fragment comprises a region representing a unit of information corresponding to one or more code elements and said code is binary.    
     
     
         3 . A method of synthesizing a double stranded nucleic acid molecule comprising at least the steps of: 
 1) generating n double stranded nucleic acid fragments, wherein at least n−2 fragments have single stranded regions at both termini and 2 fragments have single stranded regions at lest one terminus, wherein (n−1) single stranded regions are complementary to (n−1) other single stranded regions, thereby producing (n−1) complementary pairs,    2) contacting said n double stranded nucleic acid fragments, simultaneously or consecutively, to effect binding of said complementary pairs of single stranded regions, and    3) optionally ligating said complementary pairs simultaneously or consecutively to produce a nucleic acid molecule consisting of n fragments,    wherein said fragment comprises a region representing a unit of information corresponding to one or more code elements and each of said one or more code elements has the formula      (X) a ,    Wherein    X is a nucleotide A, T, G, C or a derivative thereof which allows complementary binding and may be the same or different at each position, and    a is an integer from 4 to 10,    wherein (x) a  is different from each one or more code elements.    
     
     
         4 . A method as claimed in  claim 3  wherein said code is alphanumeric.  
     
     
         5 . A method as claimed in  claim 3  wherein said code is binary.  
     
     
         6 . A method as claimed in  claim 5 , wherein said code is binary and the code elements “1” and “0” have the formulae:  
         “0”=(X) a  and “1”=(Y) b ,  
       wherein 
 (X) a  and (Y) b  are not identical,  
 X and Y are each a nucleotide A, T, G, C or a derivative thereof which allows complementary binding and may be the same or different at each position, and  
 a and b are integers from 4 to 10.  
 
     
     
         7 . A method as claimed in  claim 6  wherein in the formulae (X) a  and (Y) b , X and Y are the same at each position.  
     
     
         8 . A method as claimed in  claim 1 ,  2  or  3  wherein said fragments are each between 8 and 25 bases in length.  
     
     
         9 . A method as claimed in  claim 1 ,  2  or  3  wherein n is at least 10.  
     
     
         10 . A method of synthesizing a double stranded nucleic acid molecule comprising at least the steps of: 
 1) generating fragment chains according to the method defined in  claim 1 ,  2  or  3 ;    2) optionally generating single stranded regions at the end of said fragment chains, wherein said single stranded regions are complementary to the single stranded regions on said fragment chains thus forming complementary pairs of single stranded regions;    3) contacting said fragment chains with one another, simultaneously or consecutively, to effect binding of said complementary pairs of single stranded regions.    
     
     
         11 . A nucleic acid molecule produced according to a method as defined in  claim 1 ,  2  or  3 , or a single stranded nucleic acid molecule thereof.  
     
     
         12 . A method of identifying the code elements contained in a nucleic acid molecule prepared according to a method as defined in  claim 1 ,  2  or  3 , wherein a probe, carrying a signaling means, specific to one or more code elements, is bound to said nucleic acid molecule and a signal generated by said signalling means is detected, whereby said one or more code elements may be identified.  
     
     
         13 . A library of fragments as defined in  claim 1 ,  2  or  3 , comprising (n) m fragments, wherein n is as defined in  claim 1 ,  2  or  3  and corresponds to the length of chain that said library may produce, and m is an integer corresponding to the number of possible code elements or combinations thereof, such that fragments corresponding to all possible code elements for each position in the final chain are provided.  
     
     
         14 . A kit for synthesizing a double stranded nucleic acid molecule comprising a library as defined in  claim 13  and a ligase.