Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-27
2003-02-11
Sisson, B. L. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S091100, C435S183000, C435S287200, C436S094000, C536S023100, C536S024300
Reexamination Certificate
active
06518023
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to methods for construction physical maps of genomic DNA, and more particularly, to a method of providing high resolution physical maps using a parallel DNA sequencing technology, such as massively parallel signature sequencing (MPSS).
BACKGROUND
Physical maps of one or more large pieces of DNA, such as a genome or chromosome, consist of an ordered collection of molecular landmarks that may be used to position, or map, a smaller fragment, such as clone containing a gene of interest, within the larger structure, e.g. U.S. Department of Energy, “Primer on Molecular Genetics,” from Human Genome 1991-92 Program Report; and Los Alamos Science, 20: 112-122 (1992). An important goal of the Human Genome Project has been to provide a series of genetic and physical maps of the human genome with increasing resolution, i.e. with reduced distances in basepairs between molecular landmarks, e.g. Murray et al, Science, 265: 2049-2054 (1994); Hudson et al, Science, 270: 1945-1954 (1995); Schuler et al, Science, 274: 540-546 (1996); and so on. Such maps have great value not only in furthering our understanding of genome organization, but also as tools for helping to fill contig gaps in large-scale sequencing projects and as tools for helping to isolate disease-related genes in positional cloning projects, e.g. Rowen et al, pages 167-174, in Adams et al, editors, Automated DNA Sequencing and Analysis (Academic Press, New York, 1994); Collins, Nature Genetics, 9: 347-350 (1995); Rossiter and Caskey, Annals of Surgical Oncology, 2: 14-25 (1995); and Schuler et al (cited above). In both cases, the ability to rapidly construct high-resolution physical maps of large pieces of genomic DNA is highly desirable.
Two important approaches to genomic mapping include the identification and use of sequence tagged sites (STS's), e.g. Olson et al, Science, 245: 1434-1435 (1989); and Green et al, PCR Methods and Applications, 1: 77-90 (1991), and the construction and use of jumping and linking libraries, e.g. Collins et al, Proc. Natl. Acad. Sci., 81: 6812-6816 (1984); and Poustka and Lehrach, Trends in Genetics, 2: 174-179 (1986). The former approach makes maps highly portable and convenient, as maps consist of ordered collections of nucleotide sequences that allow application without having to acquire scarce or specialized reagents and libraries. The latter approach provides a systematic means for identifying molecular landmarks spanning large genetic distances and for ordering such landmarks via hybridization assays with members of a linking library.
Unfortunately, these approaches to mapping genomic DNA are difficult and laborious to implement. It would be highly desirable if there was an approach for constructing physical maps that combined the systematic quality of the jumping and linking libraries with the convenience and portability of the STS approach.
SUMMARY OF THE INVENTION
Accordingly, an object of my invention is to provide a method for constructing high resolution physical maps of genomic DNA.
Another object of my invention is to provide a method mapping genomic DNA by massively parallel signature sequencing of restriction fragments of the genomic DNA.
Another object of my invention is to provide a method of ordering restriction fragments by aligning matching sequences of their ends.
A further object of my invention is to provide physical maps of genomic DNA that consist of an ordered collection of nucleotide sequences spaced at an average distance of a few kilobases or less.
My invention achieves these and other objects by providing a method for constructing a physical map of a polynucleotide. In accordance with the invention, a polynucleotide is digested successively with at least two different restriction endonucleases and the ends of the restriction fragments are sequenced after each digestion. In this manner, restriction fragments having sequenced ends are produced that can be aligned by their sequences to give a physical map of the polynucleotide. Preferably, restriction fragment ends are sequenced by massively parallel signature sequencing (MPSS), or a like parallel sequencing technique.
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Gorthey LeeAnn
Lynx Therapeutics, Inc.
Powers Vincent M.
Sisson B. L.
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