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However, the tags still have an "outside-in" topology, and the fixed ~20 base tags produced by Mme I digestion are simply too short to clearly map complex genomes for use as genomic tools or assisted sequence assembly
Furthermore, fixed 20-base tags do not benefit from recent improvements in read length for next-generation short-read DNA sequencers
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[0023]The present invention provides novel and improved high throughput methods, vectors and vector components for screening and identifying fine structural changes in nucleic acid populations. The present invention includes in vitro and in vivo methods of generating a juxtaposed sequence tag (GVT) in which the two constituent members of a tag pair (GVT pair) are unique position markers separated by a defined distance and / or are markers of nucleic acid positions, It demarcates adjacent cleavage sites for one or more different restriction endonucleases along the length of a plurality of target nucleic acid molecules. The method includes: fragmenting a target nucleic acid molecule to form a target DNA insert; ligating the target DNA insert to a DNA vector or backbone to generate a circular molecule; Restriction endonuclease digestion of the target DNA insert to cleave the target DNA insert at a distance from each end of the target DNA insert, resulting in two sequence tags (GVT)...
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Abstract
A method of juxtaposing sequence tags (GVTs) that are unique positional markers along the length of a population of target nucleic acid molecules is provided, the method comprising: fragmenting the target nucleic acid molecule to form target DNA insert; ligating the target DNA insert to a DNA vector or backbone to create a circular molecule; digesting the target DNA insert endonuclease to cleave the target DNA insert at a distance from each end of the target DNA insert yielding two GVTs comprising terminal sequences of the target DNA insert attached to an undigested linear backbone; recircularizing the linear backbone with the attached GVTs to obtain a circular DNA containing a GVT-pair having two juxtaposed GVTs; and recovering the GVT-pair DNA by nucleic acid amplification or digestion with endonuclease having sites flanking the GVT-pair. Cosmid vectors are provided for creating GVT-pairs of -45- to 50-kb separation sequencable by next-generation DNA sequencers.
Description
[0001] Cross References to Related Applications [0002] This application claims priority based on: U.S. Patent Application No. 60 / 756,417, filed January 4, 2006; U.S. Patent Application No. 60 / 792,926, filed April 17, 2006; U.S. Patent Application No. 60 / 814,378, filed on July 15, 2008; U.S. Patent Application No. 61 / 129,660, filed on July 10, 2008; U.S. Patent Application No. 61 / 193,442, filed on December 1, 2008; and US Patent Application No. 11 / 954,947, filed December 12, 2007, both of which are incorporated herein by reference in their entireties. field of invention [0003] In general, the present invention relates to methods for high-throughput analysis of fine structural changes in nucleic acids. In particular, the present invention relates to novel strategies, vectors, and other components for producing ligated nucleic acid tag pairs whose constituent members have user-defined separation distances and / or are markers for nucleic acid positions along the The length of...
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