METHOD OF GENERATING NESTED SETS OF DOUBLE STRANDED DNA CIRCLES

• US 20080102466A1
• Filed: 10/25/2007
• Published: 05/01/2008
• Est. Priority Date: 10/29/2006
• Status: Active Grant

• Current Assignee: Stephen Macevicz
• Original Assignee: Stephen Macevicz
• Inventors: Macevicz, Stephen
• Granted Patent: US 7,682,791 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2527/149 Concentration of an enzyme

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.

Claims

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, the adaptor containing a recognition site for a restriction endonuclease that has a cleavage site located 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) the restriction endonuclease cleaves double stranded DNA circles to produce linear DNA molecules having ends with overhanging strands, (ii) a polishing enzyme removes overhanging strands from linear DNA molecules to produce blunt-end linear DNA molecules, and (iii) a ligase forms closed double stranded DNA circles from linear blunt-end DNA molecules.

2. The method of claim 1 further including the step of stopping one or more of said enzymatic activities and isolating said double stranded DNA circles.

3. A method of nucleic acid sequence analysis, the method comprising the steps of:

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 cleave 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.

4. A composition comprising a nested set of double stranded DNA circles, wherein each double stranded DNA circle in such set comprises a DNA fragment and at least one adaptor having a recognition site for an endonuclease having a cleavage site separate from its recognition site, the recognition site being oriented such that the cleavage site is within the DNA fragment a predetermined distance, the nested set comprising a plurality of such double stranded DNA circles such that each member of the plurality has a DNA fragment truncated by an integral number of segments, each segment having a length equivalent to the predetermined distance. from the adaptor

5. The composition of claim 4 wherein said plurality is at least three.

6. The composition of claim 5 wherein said predetermined distance is between one and four nucleotides, inclusive.

7. The composition of claim 6 wherein a majority of said truncated DNA fragments are truncated by between zero and ten said segments.

8. A method for generating a nested set of double stranded DNA circles, the method comprising the steps of:

providing one or more 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; and

circularizing the linear double stranded DNA molecules of the set to form a nested set of double stranded DNA circles.

9. The method of claim 8 wherein said primer has a 5′

-phosphate group and wherein said step of circularization is carried out by treating said linear double stranded DNA molecules with a ligase under conditions that promote self-ligation over concatenation.

10. The method of claim 9 wherein said step of circularization further includes treating said linear double stranded DNA molecules with a 5′

-exonuclease to form blunt ends thereon.

11. The method of claim 10 wherein said ligatable terminator is a 3′

-aminonucleoside triphosphate.