Koch Lab:Protocols/Unzipping constructs: Difference between revisions
(New page: ==Principle== If DNA is attached to a surface (e.g. a coverglass) at one end and to another surface (e.g. a microsphere) in the middle, the DNA can be unzipped if there is a nick between t...) |
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==Principle== | ==Principle== | ||
If DNA is attached to a surface (e.g. a coverglass) at one end and to another surface (e.g. a microsphere) in the middle, the DNA can be unzipped if there is a nick between the two attachments. This was first shown by Bockelmann, Essevaz-Roulet, and Heslot in the mid-1990s (PMID 9342340). Compared to DNA constructs for end-to-end DNA stretching (see, e.g. [[Koch_Lab:Protocols/Dig-bio_PCR|labeling DNA by PCR]] unzipping constructs are more challenging to produce. The method we describe here has a significant stretch of double-stranded DNA (dsDNA) between the first and second attachment labels (dig and biotin). A simpler construct can be made by directly hybridizing two end-labeled DNA oligos, producing a force construct. (See, e.g. [[Koch_Lab:Protocols/Fork unzipping constructs]].) | [[Image:Unzipping construct.jpg|right|thumb|'''Unzipping construct''', based on Koch et al. 2002]] | ||
If DNA is attached to a surface (e.g. a coverglass) at one end and to another surface (e.g. a microsphere) in the middle, the DNA can be unzipped if there is a nick between the two attachments. This was first shown by Bockelmann, Essevaz-Roulet, and Heslot in the mid-1990s (PMID 9342340). We describe here a versatile adaptation first described in Koch et al. 2002 (PMID 12124289). | |||
Compared to DNA constructs for end-to-end DNA stretching (see, e.g. [[Koch_Lab:Protocols/Dig-bio_PCR|labeling DNA by PCR]] unzipping constructs are more challenging to produce. The method we describe here has a significant stretch of double-stranded DNA (dsDNA) between the first and second attachment labels (dig and biotin). A simpler construct can be made by directly hybridizing two end-labeled DNA oligos, producing a force construct. (See, e.g. [[Koch_Lab:Protocols/Fork unzipping constructs]].) | |||
Revision as of 14:42, 14 June 2008
Principle
If DNA is attached to a surface (e.g. a coverglass) at one end and to another surface (e.g. a microsphere) in the middle, the DNA can be unzipped if there is a nick between the two attachments. This was first shown by Bockelmann, Essevaz-Roulet, and Heslot in the mid-1990s (PMID 9342340). We describe here a versatile adaptation first described in Koch et al. 2002 (PMID 12124289).
Compared to DNA constructs for end-to-end DNA stretching (see, e.g. labeling DNA by PCR unzipping constructs are more challenging to produce. The method we describe here has a significant stretch of double-stranded DNA (dsDNA) between the first and second attachment labels (dig and biotin). A simpler construct can be made by directly hybridizing two end-labeled DNA oligos, producing a force construct. (See, e.g. Koch_Lab:Protocols/Fork unzipping constructs.)
