Plurality of transposase adapters for dna manipulations

Abstract

Synthetic nucleic acid adaptors for transposases and methods with the adaptors and transposases, including methods for preparing DNA molecules, in vitro amplification of nucleic acids, sequencing of nucleic acids, and screening of DNA libraries for sequences of interest as well as nucleic acid delivery.

What is claimed is: 
     
         1 . An isolated synthetic nucleic acid adapter comprising:
 a first strand comprising a first sequence; and   a second strand comprising a second sequence complementary or partially complementary to the first sequence;   the first sequence and/or the second sequence comprises one or more modifications as compared to a parental recognition sequence for a transposase or the complement thereof, wherein the parental recognition sequence is selected from the group consisting of SEQ ID NOs: 1-21 and the adaptor is recognized by the transposase, and   wherein said one or more modifications being selected from the group consisting of the following:   (a) one or more modifications at the 5′ terminus of the first sequence, or at the 3′ terminus of the second sequence, or at both;   (b) one or more modified nucleotides in the first strand or the first sequence, wherein said one or more modified nucleotides impede a primer extension of said first stand;   (c) one or more phosphorothioate bonds in the second strand or the second sequence; and   (d) any combination thereof   
     
     
         2 . The adapter of  claim 1 , wherein the first sequence has at least one additional nucleotide at its 5′ terminus as compared to the parent recognition sequence, and/or the second sequence has at least one additional nucleotide at its 3′ terminus as compared to the complement of the parent recognition sequence. 
     
     
         3 . The adapter of  claim 1 , wherein the first sequence lacks at least one nucleotide at its 5′ terminus as compared to the native recognition sequence, and/or the second sequence lacks at least one nucleotide at its 3′ terminus as compared to the complement. 
     
     
         4 . The adapter of  claim 1 , wherein the first sequence and the second sequence are, respectively, SEQ ID NOs: 73 and 74, SEQ ID NOs: 75 and 76, SEQ ID NOs: 28 and 36, SEQ ID NOs: 28 and 22, SEQ ID NOs: 2 and 36, SEQ ID NOs: 2 and 74, SEQ ID NOs: 2 and 76, SEQ ID NOs: 26 and 22, SEQ ID NOs: 26 and 36, SEQ ID NOs: 26 and 39, SEQ ID NOs: 26 and 41, SEQ ID NOs: 24 and 22, SEQ ID NOs: 24 and 36, SEQ ID NOs: 24 and 39, SEQ ID NOs: 24 and 41, SEQ ID NOs: 28 and 39, SEQ ID NOs: 28 and 41, SEQ ID NOs: 2 and 39, SEQ ID NOs: 2 and 41, SEQ ID NOs: 75 and 36, or SEQ ID NOs: 73 and 36. 
     
     
         5 . The adapter of  claim 1 , wherein said one or more modified nucleotides in part (b) are selected from the group consisting of a deoxyuridine, an abasic site, a 2′OMe modified ribonucleic acid (RNA), an inverted thymidine, and any combination thereof. 
     
     
         6 . The adapter of  claim 1 , wherein the adaptor has one or more of the following features:
 (i) an inverted thymidine at the 3′ terminus of the first strand;   (ii) said one or more modified nucleotides are preceded by a phosphorothioate bond or a spacer; and   (iii)the second strand is free of said one or more modified nucleotides.   
     
     
         7 . The adapter of  claim 1 , wherein the adaptor has one or more of the following features:
 (i) one phosphorothioate bond is between the 3′ ultimate nucleotide and the 3′ pen-ultimate nucleotide of the second strand or the second sequence;   (ii) the second strand or the second sequence comprises about 1 to 18, about 2 to 15, or 9 phosphorothioate bonds;   (iii) the first strand or the first sequence is free of phosphorothioate bonds; and   (iv) the first strand is 17-80 nucleotides in length and/or the second strand is 17-80 nucleotides in length.   
     
     
         8 . The adapter of  claim 1 , wherein the adaptor has one or more of the following features:
 (i) the first strand and the second strand form a duplex of 15-30 bp in length;   (ii) the first strand and the second strand form a duplex which has a blunt end or a staggered end at the 3′ terminus of the second strand or the 5′ terminus of the first strand; and   (iii) the one or more modifications in the first or second sequence result in one or more unpaired nucleotides in the duplex.   
     
     
         9 . The adapter of  claim 1 , wherein the adaptor has one or more of the following features:
 (i) the first or second strand comprise at least one modified nucleotide selected from the group consisting of 2-Aminopurine, 2,6-Diaminopurine, 5-Bromo dU, deoxyUridine, inverted dT, inverted Dideoxy-T, dideoxy-C, 5-Methyl dC, deoxyInosine, a universal base comprising 5-Nitroindole, a 2′-O-Methyl RNA base, iso-dC, iso-dG, ribonucleotide, morpholino, a protein nucleitide analogue, a glycoic nucleotide analogue, a locked nucleotide analogue, a threose nucleotide analogue, a chain terminating nucleotide analogue, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine;   (ii) at least one nucleotide in the first or second strand is phosphorylated;   (iii) at least one nucleotide in the first or second strand comprises one or more selected from the group consisting of a modified sugar, an unnatural bond, an abasic site, a dideoxy base, a 5-methyl base, and a spacer;   (iv) at least one nucleotide in the first or second strand is a ribonucleotide;   (v) the transposase is a “cut and paste” transposase, Vibrio Harveyi transposase or hyperactive Tn5 transposase;   (vi) the second strand further comprises a tag sequence 5′ to the second sequence or the first strand further comprises a tag sequence 3′ to the first sequence; and   (vii) the tag sequence comprises a degenerate base region.   
     
     
         10 . A set of isolated synthetic nucleic acid adapters, comprising (i) a first isolated synthetic nucleic acid adapter of  claim 1 , and (ii) a second isolated synthetic nucleic acid adapter of  claim 1 , wherein the first adapter and the second adapter have at least one different modification as compared to the parent recognition sequence or the complement thereof. 
     
     
         11 . A transposase complex comprising one or more transposase molecules and one or more adapters of  claim 1 . 
     
     
         12 . An in vitro method for fragmenting a target DNA molecule, comprising: contacting a target DNA molecule with a transposase complex of  claim 1  to form a reaction mixture; and incubating the reaction mixture under conditions for carrying out a transposition reaction. 
     
     
         13 . The method of  claim 12 , wherein the target DNA molecule is obtained from a sample comprising 1-10 or 1-3 cells. 
     
     
         14 . A method for preparing an assay sample for sequencing or microarray analysis of a target DNA molecule, comprising:
 contacting a target DNA molecule with a complex comprising the isolated synthetic nucleic acid adapter of  claim 1  and a transposase that binds to the adapter to form a reaction mixture;   incubating the reaction mixture under conditions for carrying out a transposition reaction and thereby generating a cleaved DNA product, and   amplifying the cleaved DNA product.   
     
     
         15 . The method of  claim 14 , wherein the amplifying step is conducted without prior removing of the adaptor or separating the cleaved DNA product from the reaction mixture. 
     
     
         16 . A method of DNA sample preparation for microarrays, comprising contacting a sample DNA molecule with a complex comprising the isolated synthetic nucleic acid adapter of  claim 1  and a transposase that binds to the adapter to form a reaction mixture; and incubating the reaction mixture under conditions for carrying out a transposition reaction to generate DNA fragments of the sample DNA molecule, wherein said adapter comprises an oligonucleotide tag and the DNA fragments are tagged at both ends with the oligonucleotide tag. 
     
     
         17 . The method of  claim 16 , further comprising amplifying the DNA fragments using primers complementary to the oligonucleotide tag. 
     
     
         18 . The method of  claim 16 , wherein said tag is used as a landing site for a primer complementary to the tag. 
     
     
         19 . The method of  claim 17 , wherein the primer or primers are extended in a polymerase reaction using a dNTP mixture comprising at least one dNTP labeled with a fluorophore. 
     
     
         20 . The transposase complex of  claim 11 , wherein the complex is bound to a solid support.