Method for modifying a template double stranded polynucleotide

Abstract

The invention relates to a method for modifying a template double stranded polynucleotide, especially for characterisation using nanopore sequencing. The method produces from the template a plurality of modified double stranded polynucleotides. These modified polynucleotides can then be characterised.

1 . A method for modifying a template double stranded polynucleotide, comprising:
 (a) contacting the template polynucleotide with a MuA transposase and a population of double stranded MuA substrates each comprising an overhang at one or both ends of one strand such that the transposase fragments the template polynucleotide and ligates a substrate to one or both ends of the double stranded fragments and thereby producing a plurality of fragment/substrate constructs; and   (b) using a translocase to remove the MuA transposases from the constructs and thereby producing a plurality of modified double stranded polynucleotides.   
     
     
         2 . A method according to  claim 1 , wherein the translocase is contacted with the constructs after they are created by the MuA transposase. 
     
     
         3 . A method according to  claim 1 , wherein the translocase is bound to the substrates before the substrates are contacted with the template polynucleotide. 
     
     
         4 . A method according to  claim 1 , wherein the translocase is a helicase. 
     
     
         5 . A method according to  claim 4 , wherein the helicase is from superfamily 1 or superfamily 2; optionally wherein the helicase is a member of one of the following families: Pif1-like, Upf1-like, UvrD/Rep, Ski-like, Rad3/XPD, NS3/NPH-II, DEAD, DEAH/RHA, RecG-like, REcQ-like, T1R-like, Swi/Snf-like and Rig-I-like; or wherein the helicase is a UvrD helicase, a Hel308 helicase, a TraI helicase, a TraI subgroup helicase, an XPD helicase or a Dda helicase. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . A method according to  claim 4 , wherein the helicase is a Hel308 helicase; optionally wherein the Hel308 helicase is Hel308 Mbu (E284C/S615C)-bismaleimidePEG11 (SEQ ID NO: 10 with mutations E284C/S615C connected by a bismaleimidePEG11 linker). 
     
     
         9 . (canceled) 
     
     
         10 . A method according to  claim 1 , wherein the translocase is a strippase; optionally wherein the strippase is the INO80 chromatin remodeling complex or a FtsK/SpoIIIE transporter. 
     
     
         11 . (canceled) 
     
     
         12 . A method according to  claim 1 , wherein the two strands of each construct are linked at one end by a hairpin loop. 
     
     
         13 . A method according to  claim 1 , wherein the method further comprises attaching molecular brakes to the other strands in the substrates. 
     
     
         14 . A method according to  claim 13 , wherein the molecular brakes are attached to the other strands in the substrates before they are contacted with the template polynucleotide and the MuA transposase. 
     
     
         15 . A method according to  claim 13 , wherein the molecular brakes are attached to the other strands from the substrates remaining in the constructs after they are created by the MuA transposase. 
     
     
         16 . A method according to  claim 13 , wherein the molecular brakes are bound to Y adaptors comprising a leader sequence and/or one or more anchors capable of coupling the adaptor to a membrane and the Y adaptors are attached to the other strands in step (c). 
     
     
         17 . A method according to  claim 13 , wherein the molecular brakes are derived from a polymerase, a helicase or an exonuclease. 
     
     
         18 . (canceled) 
     
     
         19 . A population of double stranded MuA substrates for modifying a template polynucleotide, wherein each substrate comprises an overhang at one or both ends of one strand and a translocase bound to an overhang. 
     
     
         20 . A plurality of polynucleotides modified using a method according to  claim 1 . 
     
     
         21 . A method of characterising at least one polynucleotide modified using a method according to  claim 1 , comprising:
 a) contacting the modified polynucleotide with a transmembrane pore such that at least one strand of the polynucleotide moves through the pore; and   b) taking one or more measurements which are indicative of one or more characteristics of the polynucleotide as the at least one strand moves with respect to the pore and thereby characterising the modified polynucleotide.   
     
     
         22 . A method of characterising a template polynucleotide, comprising:
 a) modifying the template polynucleotide using a method according to  claim 1  to produce a plurality of modified polynucleotides;   b) contacting each modified polynucleotide with a transmembrane pore such that at least one strand of each polynucleotide moves through the pore; and   c) taking one or more measurements which are indicative of one or more characteristics of the polynucleotide as the at least one strand of each polynucleotide moves with respect to the pore and thereby characterising the template polynucleotide.   
     
     
         23 . A method according to  claim 21 , wherein the one or more characteristics are selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether or not the polynucleotide is modified. 
     
     
         24 . A method according to  claim 21 , wherein the method comprises measuring the current passing through the pore as the at least one strand or each polynucleotide moves with respect to the pore. 
     
     
         25 . A kit for modifying a template polynucleotide comprising (a) a population of MuA substrates as defined in  claim 1 , (b) a MuA transposase and (c) a translocase; optionally wherein the kit further comprises a polynucleotide protein and/or a Y adaptor comprising a leader sequence and/or one or more anchors capable of coupling the adaptor to a membrane. 
     
     
         26 . (canceled)