Adaptors for nucleic acid constructs in transmembrane sequencing

Abstract

The invention relates to adaptors for sequencing nucleic acids. The adaptors may be used to generate single stranded constructs of nucleic acid for sequencing purposes. Such constructs may contain both strands from a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) template. The invention also relates to the constructs generated using the adaptors, methods of making the adaptors and constructs, as well as methods of sequencing double stranded nucleic acids.

35 . A method of preparing a nucleic acid construct for sequencing comprising:
 (a) providing a double stranded nucleic acid template;   (b) covalently attaching a first hairpin adaptor at one end of the double stranded nucleic acid template and a second hairpin adaptor the other end of the double stranded nucleic acid template to form a circular nucleic acid construct;   
       wherein said first hairpin adaptor and said second hairpin adaptor are differentially selectable. 
     
     
         36 . The method of  claim 35 , further comprising the step of determining the sequence of the double stranded nucleic acid construct. 
     
     
         37 . The method of  claim 36 , wherein the sequence of the double stranded nucleic acid construct is determined by a sequencing by synthesis method. 
     
     
         38 . The method of  claim 35 , further comprising the step of binding the nucleic acid construct to a surface. 
     
     
         39 . The method of  claim 35 , wherein the first hairpin adaptor and the second adaptor each comprises a selectable binding moiety. 
     
     
         40 . The method of  claim 39 , wherein at least one of the selectable binding moieties allows binding of the nucleic acid construct to a surface. 
     
     
         41 . The method of  claim 39 , wherein the selectable binding moiety is a selectable nucleic acid sequence. 
     
     
         42 . The method of  claim 39 , wherein the selectable binding moiety is a nucleic acid binding protein. 
     
     
         43 . The method of  claim 39 , wherein the selectable binding moiety is biotin. 
     
     
         44 . The method of  claim 39 , wherein the selective binding moiety is comprised within a hairpin loop of the hairpin adaptor. 
     
     
         45 . The method of  claim 35 , wherein at least one of the first or second hairpin adaptors comprises a sticky end of either a 5′ or 3′ overhang. 
     
     
         46 . The method of  claim 35 , wherein each hairpin adaptor comprises a region of double stranded nucleic acid formed by hybridization between two separate regions of a single stranded nucleic acid and the two regions of the single stranded nucleic acid are the same type of nucleic acid or are different types of nucleic acid. 
     
     
         47 . The method of  claim 35 , wherein step (a) further comprises randomly fragmenting template nucleic acid. 
     
     
         48 . The method of  claim 35 , wherein the sequence of the double stranded nucleic acid is known or can be predicted. 
     
     
         49 . The method of  claim 35 , wherein the sequence of the double stranded nucleic acid is unknown. 
     
     
         50 . A pair of adaptors for sequencing nucleic acids, wherein each of the adaptors of the same pair comprises a hairpin loop and each adaptor is differentially selectable from the other adaptor of the same pair. 
     
     
         51 . The pair of adaptors of  claim 50 , wherein the first hairpin adaptor and the second adaptor of each pair each comprise a selectable binding moiety. 
     
     
         52 . The pair of adaptors of  claim 51 , wherein the selectable binding moieties are capable of facilitating construction and purification of a nucleic acid for sequencing by binding to a surface. 
     
     
         53 . A circular nucleic acid construct for use as a sequencing template comprising a double stranded nucleic acid template and a pair of adaptors of  claim 50 , wherein a first adaptor is covalently attached at one end of the double stranded nucleic acid and a second adaptor is covalently attached at the other end of the double stranded nucleic acid.