Methods and Oligonucleotide Designs for Insertion of Multiple Adaptors into Library Constructs
First Claim
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1. A method for selectively activating a recognition site for a Type IIs restriction endonuclease in a nucleic acid sequence, the method comprising:
- (a) providing a nucleic acid sequence comprising first and second recognition sites for a Type IIs restriction endonuclease;
(b) amplifying the nucleic acid sequence using a uracil-containing primer that has a sequence that is complementary to the first recognition site, thereby producing an amplified nucleic acid sequence comprising a first recognition site for a Type IIs restriction endonuclease comprising one or more uracils at or near the first recognition site, and a second recognition site for a Type IIs restriction endonuclease;
(c) degrading said one or more uracils at or near the first recognition site, thereby producing a single-stranded region in the first recognition site and protecting the first recognition site from nicking by a nickase that nicks unprotected recognition sites for the Type IIs restriction endonuclease;
(f) nicking the second recognition site with the nickase, thereby inhibiting digestion of the nucleic acid sequence by the Type IIs restriction endonuclease resulting from recognition of the second recognition site; and
(g) making the single-stranded region double-stranded such that the Type IIs restriction endonuclease can recognize the first recognition site and digest the nucleic acid sequence.
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Abstract
Aspects described and claimed herein provide methods to insert multiple DNA adaptors into a population of circular target DNAs at defined positions and orientations with respect to one another. The resulting multi-adaptor constructs are then used in massively-parallel nucleic acid sequencing techniques.
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Citations
18 Claims
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1. A method for selectively activating a recognition site for a Type IIs restriction endonuclease in a nucleic acid sequence, the method comprising:
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(a) providing a nucleic acid sequence comprising first and second recognition sites for a Type IIs restriction endonuclease; (b) amplifying the nucleic acid sequence using a uracil-containing primer that has a sequence that is complementary to the first recognition site, thereby producing an amplified nucleic acid sequence comprising a first recognition site for a Type IIs restriction endonuclease comprising one or more uracils at or near the first recognition site, and a second recognition site for a Type IIs restriction endonuclease; (c) degrading said one or more uracils at or near the first recognition site, thereby producing a single-stranded region in the first recognition site and protecting the first recognition site from nicking by a nickase that nicks unprotected recognition sites for the Type IIs restriction endonuclease; (f) nicking the second recognition site with the nickase, thereby inhibiting digestion of the nucleic acid sequence by the Type IIs restriction endonuclease resulting from recognition of the second recognition site; and (g) making the single-stranded region double-stranded such that the Type IIs restriction endonuclease can recognize the first recognition site and digest the nucleic acid sequence.
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2. A method of positioning a second adaptor with respect to a first adaptor in a nucleic acid template construct, said method comprising:
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(a) providing a first linear construct, wherein said first linear construct comprises a target nucleic acid and a first adaptor, and wherein said first adaptor comprises a first recognition site for a first Type IIs restriction endonuclease; (b) protecting said first recognition site from inactivation; (c) inactivating unprotected restriction endonuclease recognition sites, if any, in said first linear construct; (d) circularizing said first linear construct to form a first circular construct; (e) applying said first Type IIs restriction endonuclease to said first circular construct to form a second linear construct, wherein said second linear construct comprises said first adaptor inserted within said target nucleic acid; (f) ligating a second adaptor to said second linear construct to form said nucleic acid template construct, wherein said second adaptor comprises a second recognition site for a second Type IIs restriction endonuclease; thereby positioning said second adaptor with respect to said first adaptor in said nucleic acid template construct. - View Dependent Claims (3, 4, 5, 6, 7, 14, 15, 16, 17, 18)
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8. A method of making a library of circular nucleic acid templates each comprising a target nucleic acid sequence and at least two adaptors, said method comprising:
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(a) providing fragments of genomic nucleic acid; (b) adding a first arm of a first adaptor to one terminus of a plurality of said fragments; (c) adding a second arm of a first adaptor to the other terminus of said plurality of said fragments to form first linear constructs, wherein said first and second arms of said first adaptor, when ligated, form said first adaptor and produce a first recognition site for a first Type IIs restriction endonuclease; (d) protecting said first recognition site in said first linear constructs from inactivation; (e) inactivating any unprotected first recognition sites present in said first linear constructs; (f) circularizing said first linear constructs by ligating said first and second adaptor arms to form first circular constructs; (g) cleaving said first circular constructs with said first Type IIs restriction endonuclease to form second linear constructs comprising said first adaptor inserted within said target nucleic acid, wherein said first Type IIs restriction endonuclease binds to said protected first recognition site and cleaves at a position in said first circular constructs outside of said first adaptor; (h) adding a first arm of a second adaptor to one terminus of said plurality of said second linear constructs; (i) adding a second arm of a second adaptor to the other terminus of said plurality of said fragments to form second linear constructs, wherein said first and second arms of said second adaptor, when ligated, form said second adaptor and form a second Type IIs recognition site; (j) circularizing said second linear constructs by ligating said first and second adaptor arms of said second adaptor to form second circular constructs, thereby making said library of circular nucleic acid templates. - View Dependent Claims (9, 10, 11, 12, 13)
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Specification