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.

1 . An adaptor for sequencing nucleic acids, which comprises a region of double stranded nucleic acid, wherein at least one end of the region forms one half of a palindromic cleavage site and wherein the adaptor is differentially selectable from another adaptor. 
     
     
         2 . An adaptor according to  claim 1 , wherein the region is formed by hybridization between two separated regions of a single stranded nucleic acid and the adaptor comprises a hairpin loop. 
     
     
         3 . An adaptor according to  claim 1 , wherein the one half of the palindromic cleavage site comprises blunt ends, optionally wherein the palindromic cleavage site is a restriction endonuclease recognition site. 
     
     
         4 . (canceled) 
     
     
         5 . An adaptor according to  claim 1 , wherein the adaptor can be separated from another adaptor by differential binding. 
     
     
         6 . An adaptor according to  claim 1 , wherein the adaptor comprises a selectable binding moiety, optionally wherein the selectable binding moiety is biotin or a selectable nucleic acid sequence. 
     
     
         7 . (canceled) 
     
     
         8 . An adaptor according to  claim 1 , wherein the adaptor comprises a nucleic acid sequence that allows identification of the adaptor. 
     
     
         9 . (canceled) 
     
     
         10 . A pair of adaptors comprising a Type I adaptor and a Type II adaptor, wherein each type of adaptor in the pair is differentially selectable from the other type and wherein a complete palindromic cleavage site is formed if any combination of the two types of adaptor are ligated to one another,
 wherein the Type I adaptor comprises a region of double stranded nucleic acid, wherein at least one end of the region forms one half of a palindromic cleavage site and wherein the adaptor is differentially selectable from another adaptor, and the region is formed by hybridization between two separated regions of a single stranded nucleic acid and the adaptor comprises a hairpin loop; and   wherein the Type II adaptor comprises a region of double stranded nucleic acid, wherein at least one end of the region forms one half of a palindromic cleavage site and wherein the adaptor is differentially selectable from another adaptor.   
     
     
         11 .- 12 . (canceled) 
     
     
         13 . A pair of adaptors according to  claim 10 , wherein the Type I adaptor can be separated from the Type II adaptor by differential binding optionally wherein the Type I adaptor comprises a different selectable binding moiety from the Type II adaptor. 
     
     
         14 . (canceled) 
     
     
         15 . A pair of adaptors according to  claim 10 , wherein the Type I adaptor is not itself capable of being cleaved or nicked and the Type II adaptor is itself capable of being cleaved or nicked. 
     
     
         16 . A pair of adaptors according to  claim 10 , wherein the Type I adaptor comprises a nucleic acid sequence that allows identification of the adaptor. 
     
     
         17 . (canceled) 
     
     
         18 . A nucleic acid construct for use as a sequencing template comprising a double stranded nucleic acid ligated to at least one adaptor according to  claim 1 . 
     
     
         19 . A single stranded nucleic acid construct for use as a sequencing template comprising two strands of nucleic acid covalently linked via an adaptor according to  claim 2 . 
     
     
         20 . A circular nucleic acid construct for use as a sequencing template comprising two strands of nucleic acid covalently linked at each end via an adaptor according to  claim 2 . 
     
     
         21 .- 23 . (canceled) 
     
     
         24 . A method for preparing a nucleic acid construct, comprising:
 (a) contacting at least one adaptor according to  claim 1  with two strands of nucleic acid under conditions which allow ligation between the adaptor(s) and the strands; and   (b) allowing the adaptor to ligate to the two strands and thereby preparing a nucleic acid construct.   
     
     
         25 . A method for preparing a single stranded nucleic acid construct; comprising:
 (a) contacting an adaptor according to  claim 2  with two strands of nucleic acid under conditions which allow ligation between the adaptor and the strands;   (b) allowing the adaptor to covalently link the two strands; and   (c) denaturing the covalently linked construct and thereby preparing a single stranded nucleic acid construct.   
     
     
         26 . A method for preparing a circular nucleic acid construct, comprising:
 (a) contacting at least two adaptors according to  claim 2  with two strands of nucleic acid under conditions which allow ligation between the adaptors and strands; and   (b) allowing an adaptor to covalently link the two strands at each end and thereby preparing a circular nucleic acid construct.   
     
     
         27 . A method for preparing a sequence construct, comprising:
 (a) providing double stranded nucleic acid;   (b) contacting the double stranded nucleic acid with a pair of Type I and Type II adaptors according to  claim 15  under conditions which allow the adaptors to ligate to the nucleic acid;   (c) contacting the ligated products with a surface that specifically binds the Type II adaptors and removing any unbound products;   (d) contacting the surface with an enzyme that recognises the complete palindromic cleavage site and removing any unbound products;   (e) cleaving the Type II adaptors;   (f) contacting the soluble products produced in step (e) with a surface that specifically binds the Type I adaptors and removing any unbound products; and   (g) releasing from the surface the products remaining following step (f) and thereby producing a sequencing construct.   
     
     
         28 .- 29 . (canceled) 
     
     
         30 . A method of sequencing double stranded nucleic acid, comprising:
 (a) carrying out a method according to  claim 25 ;   (b) denaturing the construct, if necessary, to form a single stranded construct; and   (c) sequencing the single stranded construct and thereby sequencing the double stranded nucleic acid, optionally wherein the double stranded nucleic acid contains or is suspected of containing methylcytosine.   
     
     
         31 . A method according to  claim 30 , wherein the sequencing in step (c) is carried out by a method comprising:
 (i) contacting the construct with a transmembrane pore having an exonuclease and a molecular adaptor covalently attached thereto so that the exonuclease digests an individual nucleotide from one end of the construct;   (ii) contacting the nucleotide with the pore so that the nucleotide interacts with the molecular adaptor;   (iii) measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide; and   (iv) repeating steps (i) to (iii) at the same end of the construct and thereby determining the sequence of the construct.   
     
     
         32 . A method according to  claim 30 , wherein the sequencing in step (c) is carried out by a method comprising:
 (i) contacting the construct with a transmembrane pore having a nucleic acid handling enzyme covalently attached thereto so that the enzyme pushes or pulls the construct through the pore and a proportion of the nucleotides in the construct interacts with the pore; and   (ii) measuring the current passing through the pore during each interaction and thereby determining the sequence of the construct.   
     
     
         33 .- 34 . (canceled)