Methods for nucleic acid mapping and identification of fine-structural-variations in nucleic acids

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] This application is a divisional application. The filing date of the original application is July 9, 2009. The application number is 200980135935.8 (PCT / CN2009 / 000777). method". [0002] Cross References to Related Applications [0003] 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 entirety. field of invention [0004] 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 comp...

AI Technical Summary

Problems solved by technology

However, the tags still have an "outside-in" topology, and the fixed ~20 base tags produced by MmeI 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