Use of site-specific nicking endonucleases to create single-stranded regions and applications thereof
First Claim
1. A method for creating a target single-stranded region in double-stranded DNA comprising:
- (a) nicking at least one site bordering the target region in double-stranded DNA with at least one site-specific nicking endonuclease; and
(b) subjecting the nicked DNA to conditions where the target region is selectively denatured.
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Accused Products
Abstract
The present invention relates to the use of site-specific nucleic acid nicking enzymes to create single-stranded regions in duplex nucleic acids. Such single-stranded regions can take the form of gaps interior to the duplex, or terminal single-stranded regions. Single-stranded termini can be crafted to allow linkage of various elements via base-pairing with elements containing a complementary single-stranded region. This joining is useful, for example, in an ordered, oriented assembly of DNA modules to create cloning or expression vectors. This joining is also useful in attaching detection probes and purifying DNA molecules containing the single-stranded region. Gaps are useful in similar applications, including attaching detection or purification probes.
54 Citations
21 Claims
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1. A method for creating a target single-stranded region in double-stranded DNA comprising:
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(a) nicking at least one site bordering the target region in double-stranded DNA with at least one site-specific nicking endonuclease; and
(b) subjecting the nicked DNA to conditions where the target region is selectively denatured. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21)
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19. A method for assembling a vector with multiple, interchangeable parts comprising element sets containing:
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(a) a replication origin and associated control sequences; and
(b) a selectable marker; and
(c) one or more of the following element sets;
(i) promoters and associated control elements;
(ii) coding sequences allowing production of fusion proteins;
(iii) transcription terminators;
(iv) regulatory proteins; and
(v) gene coding regions;
wherein each element set has unique common cohesive termini, allowing joining of elements within an ordered assembly, and interchange of elements in each set in the assembly.
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Specification