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 - 34 . (canceled) 
     
     
         35 . A method of sequencing a double stranded nucleic acid template, comprising:
 (a) providing a construct in which the two strands of the double stranded nucleic acid template are covalently linked via an adaptor, wherein the adaptor comprises a region of double stranded nucleic acid which is formed by hybridization between two separate regions of a single stranded nucleic acid and comprises a hairpin loop;   (b) denaturing the construct to form a single stranded construct;   (c) contacting the construct with a transmembrane pore and a nucleic acid handling enzyme so that the enzyme provides controlled and stepwise translocation of the construct through the pore and a proportion of the nucleotides in the construct interacts with the pore; and   (d) measuring the current passing through the pore during each interaction and thereby determining the sequence of the double stranded nucleic acid template.   
     
     
         36 . A method of sequencing a double stranded nucleic acid template comprising:
 (a) providing a construct in which the two strands of the double stranded nucleic acid template are covalently linked via an adaptor, wherein the adaptor comprises a region of double stranded nucleic acid which is formed by hybridization between two separate regions of a single stranded nucleic acid and comprises a hairpin loop;   (b) denaturing the construct to form a single stranded construct;   (c) 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;   (d) contacting the nucleotide with the pore so that the nucleotide interacts with the molecular adaptor;   (e) measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide; and   (f) repeating steps (c) to (e) at the same end of the construct and thereby determining the sequence of the double stranded nucleic acid template.   
     
     
         37 . A method according to  claim 35  or  36 , wherein the construct is provided by a method comprising:
 (a) contacting the adaptor with the two strands of the double stranded nucleic acid template under conditions which allow ligation between the adaptor and the two strands; and 
 (b) allowing the adaptor to covalently link the two strands. 
 
     
     
         38 . A method according to  claim 35 , wherein the nucleic acid handling enzyme is a polymerase, exonuclease, helicase, topoisomerase or gyrase. 
     
     
         39 . A method according to  claim 35  or  36 , wherein the transmembrane pore is a protein pore. 
     
     
         40 . A method according to  claim 35  or  36 , wherein the transmembrane pore is a protein pore derived from α-hemolysin or MspA. 
     
     
         41 . A method according to  claim 35  or  36 , wherein the membrane is an artificial membrane. 
     
     
         42 . A method according to  claim 35  or  36 , wherein the membrane is a lipid bilayer. 
     
     
         43 . A method according to  claim 35  or  36 , wherein the double stranded nucleic acid template is double stranded DNA (dsDNA) or double stranded RNA (dsRNA). 
     
     
         44 . A method according to  claim 35  or  36 , wherein the double stranded nucleic acid template contains or is suspected of containing methylcytosine. 
     
     
         45 . A method according to  claim 35  or  36 , wherein in step (b) the construct is chemically or thermally dissociated into a single strand. 
     
     
         46 . A method according to  claim 35 , wherein the nucleic acid handling enzyme is covalently attached to the transmembrane pore. 
     
     
         47 . A method according to  claim 35  or  36 , wherein the adaptor is differentially selectable from another adaptor on the basis of differential or selective binding to a surface. 
     
     
         48 . A method according to  claim 35  or  36 , wherein the two separate regions of the single stranded nucleic acid are the same type of nucleic acid or different types of nucleic acid. 
     
     
         49 . An adaptor for sequencing nucleic acids, which comprises a region of double stranded nucleic acid, wherein the adaptor is differentially selectable from another adaptor on the basis of differential or selective binding to a surface and wherein the region of double stranded nucleic acid is formed by hybridization between two separate regions of a single stranded nucleic acid and the adaptor comprises a hairpin loop. 
     
     
         50 . An adaptor according to  claim 49 , wherein the adaptor comprises a selectable binding moiety. 
     
     
         51 . An adaptor according to  claim 50 , wherein the selectable binding moiety is biotin or a selectable nucleic acid sequence. 
     
     
         52 . An adaptor according to  claim 49 , wherein the adaptor comprises a nucleic acid sequence that allows identification of the adaptor. 
     
     
         53 . An adaptor according to  claim 49 , wherein the adaptor is itself capable of being cleaved or nicked. 
     
     
         54 . An adaptor according to  claim 49 , wherein the two separate regions of the single stranded nucleic acid are the same type of nucleic acid or different types of nucleic acid. 
     
     
         55 . A nucleic acid construct for use as a sequencing template comprising a double stranded nucleic acid template ligated to at least one adaptor according to  claim 49 . 
     
     
         56 . A single stranded nucleic acid construct for use as a sequencing template comprising two strands of a nucleic acid template covalently linked via an adaptor according to  claim 49 .