Method of generating nested sets of double stranded DNA circles
First Claim
1. A method for generating a nested set of double stranded DNA circles, the method comprising the steps of:
- providing one or more double stranded DNA circles each comprising an adaptor and a DNA fragment separate from the adaptor, the adaptor containing a recognition site for a restriction endonuclease that has a cleavage site located a predetermined distance within the DNA fragment and that produces cleavage products having overhanging strands; and
combining in a reaction mixture the one or more double stranded DNA circles under conditions such that the following enzymatic activities are present;
(i) a restriction endonuclease activity recognizing the restriction site in the adaptor, which cleaves the DNA fragment of the double stranded DNA circles to produce linear DNA molecules having ends with overhanging strands, (ii) a polishing enzyme activity that removes overhanging strands from linear DNA molecules to produce blunt-end linear DNA molecules, and (iii) a ligase activity that forms closed double stranded DNA circles from linear blunt-end DNA molecules, whereby a cyclical reaction takes place to generate a nested set of shortened double stranded DNA circles in the reaction mixture.
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Abstract
The invention provides a method of generating nested sets of double stranded DNA (dsDNA) circles that may be used as size ladders in nucleic acid separations and as templates in DNA sequencing operations. In one aspect, the invention provides methods for generating nested sets of double stranded DNA circles in a self-sustaining enzymatic reaction comprising the activities of at least one endonuclease, at least one single stranded exonuclease, and at least one ligase. In another embodiment, such nested sets are generated from linear dsDNA fragments having ligatable terminators that are self-ligated to form corresponding dsDNA circles.
10 Citations
10 Claims
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1. A method for generating a nested set of double stranded DNA circles, the method comprising the steps of:
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providing one or more double stranded DNA circles each comprising an adaptor and a DNA fragment separate from the adaptor, the adaptor containing a recognition site for a restriction endonuclease that has a cleavage site located a predetermined distance within the DNA fragment and that produces cleavage products having overhanging strands; and combining in a reaction mixture the one or more double stranded DNA circles under conditions such that the following enzymatic activities are present;
(i) a restriction endonuclease activity recognizing the restriction site in the adaptor, which cleaves the DNA fragment of the double stranded DNA circles to produce linear DNA molecules having ends with overhanging strands, (ii) a polishing enzyme activity that removes overhanging strands from linear DNA molecules to produce blunt-end linear DNA molecules, and (iii) a ligase activity that forms closed double stranded DNA circles from linear blunt-end DNA molecules, whereby a cyclical reaction takes place to generate a nested set of shortened double stranded DNA circles in the reaction mixture. - View Dependent Claims (2)
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3. A method of nucleic acid sequence analysis, the method comprising the steps of:
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providing a plurality of first double stranded DNA circles, each such DNA circle having a DNA fragment and an adaptor the adaptor having at least two type IIs restriction sites oriented so that mate-pairs are produced upon cleavage with type IIs restriction endonucleases recognizing such sites and the adaptor having a third type IIs restriction site oriented so that a third type IIs restriction endonuclease recognizing such third site cleaves each double stranded DNA circle at a cleavage site proximal to the adaptor relative to that of a type IIs restriction endonuclease that produces a mate-pair; cleaving a portion of the plurality of first double stranded DNA circles with one of the at least two type IIs restriction endonucleases and the third type IIs restriction endonuclease to produce a small linear double stranded DNA fragment and a large linear double stranded DNA fragment, each small linear DNA fragment comprising a segment adjacent to the adaptor of its first double stranded DNA circle; circularizing the large linear DNA fragments to produce second double stranded DNA circles; generating mate-pairs from the first and second double stranded DNA circles; and determining the nucleotide sequences of the mate-pairs adjacent to the adaptors. - View Dependent Claims (9, 10)
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4. A method for generating a nested set of double stranded DNA circles, the method comprising the steps of:
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providing one or morn single stranded DNA sequences each having a primer binding site; extending a primer annealed to the primer binding site by a nucleic acid polymerase in the presence of a ligatable terminator to form a nested set of linear double stranded DNA molecules; treating the linear double stranded DNA molecules with a 5′
-exonuclease to form blunt ends thereon; andcircularizing the linear double stranded DNA molecules of the set to form a nested set of double stranded DNA circles. - View Dependent Claims (5, 6, 7, 8)
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Specification