Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-04-14
2002-05-21
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S440000, C435S471000, C435S320100
Reexamination Certificate
active
06391642
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to methods of efficiently producing yeast artificial chromosomes and to methods of efficiently and selectively cloning yeast artificial chromosomes from mixed population of nucleic acids. The invention also relates to a method of selectively cloning a specific nucleic acid. Also included in the present invention are vectors used in these methods and products formed using these methods.
2. Background Art
A critical step in the characterization of large genomes, including that of humans, has been the cloning of large chromosomal fragments. This has been fulfilled through the development of artificial chromosomes in the yeast
Saccharomyces cerevisiae
(YACs) and has led to the large scale physical map of the human genome derived from several YAC libraries (Burke, et al.,
Science
236, 806-812 (1987) and Guyer, et al.,
Proc. Natl. Acad. Sci. USA
92, 10841-10848 (1995)).
Because of their large size, YACs have proven essential in genome mapping of many organisms. However, artifacts such as chimeras and deletions can limit their use (Kouprina, et al.,
Genomics
21, 7-17 (1994), Green, et al, Genonics 11,658-669 (1991), Wada, et al., (1994)
Nucleic Acids Res.
22, 1561-1554 (1994), and Schlessinger, et al.,
Genomics
11, 783-794 (1991)). These artifacts may result from in vitro DNA manipulation resulting in the DNA becoming broken or nicked. Up to 50% of YACs in libraries are represented by chimeric clones containing noncontiguous fragments of DNA; the chimeric YACs could result from in vitro DNA ligation as well as from in vivo recombination between co-penetrating DNA molecules (Green, et al.,
Genomics
11, 658-669 (1991), Wada, et al.,
Nucleic Acids Res.
22, 1561-1554 (1994), and Larionov, et al.,
Nucleic Acids Res.
22, 4154-4161 (1994)). In addition internal deletions can occur during propagation of some YACs (Neil, et al.,
Nucleic Acids Res.
18, 1421-1428 (1990) and Kouprina, et al.,
Genomics
21, 7-17 (1994)), probably from recombinational interactions between repeated sequences.
Improved fidelity of cloned DNA can be accomplished through the generation of artificial chromosomes in bacteria (BACs and PACs) (Shizuya, et al.,
Proc. Natl. Acad. Sci. USA
89, 8794-8797 (1992) and Shepherd, et al.,
Genetic Engineering Ed.
by J. K. Setlow, 16, 213-228 (1994)). However, the average size of these artificial chromosomes is much smaller (<150 kb) than can be attained with YACs where megabase YACs have been utilized for mapping. The lack of chimeras and the stability of BACs during propagation have contributed to their utility, especially in conjunction with physical mapping information obtained with YACs. They have been especially useful for closure of some of the gaps between contigs generated using large YAC clones.
It has become apparent that the ability to isolate specific chromosomal regions would greatly benefit positional cloning and studies of various human diseases as well as fill the gaps in existing maps. Human chromosome and subchromosome-specific libraries can be prepared from either flow-sorted chromosomes or from genomic DNA of somatic hybrid cell lines containing a single human chromosome (McCormick, et al.,
Genomics
18, 553-558 (1993)). Despite the advantages, the generation of libraries from flow-sorted chromosomes is a laborious process due to the difficulty in purifying sufficient quantities of chromosomes. Because of this, many chromosome-specific libraries have been generated by cloning directly from monochromosomal hybrid lines, with the clones being screened for human inserts by hybridization. (Gingrich, et al.,
Genomics
15, 228-230 (1993)).
This invention overcomes problems in the art by providing an alternative approach for cloning human DNA into yeast as large linear YACs that omits the in vitro ligation step. The approach is based on transformation-associated recombination (TAR). In one embodiment the TAR occurs between a repeat within transformed DNA fragments (such as an Alu or LINE) and a repeat sequence on a co-transformed linearized plasmid that also contains a yeast centromere and a telomere. Using this new YAC construction technique, we have successfully made YACs containing human DNA from human chromosomes without in vitro enzymatic treatment to the DNA. We have also successfully used this technique to selectively clone specific DNA from a background of mixed DNAs. This technique, therefore, has a tremendous utility of being able to selectively isolate DNAs from hybrid cell or other populations where the DNAs are from mixed origins.
Based on this new TAR cloning procedure, we have, in a specific embodiment, also exploited the use of TAR cloning centromere vectors that have human DNA repeats at both ends in order to generate large circular YACs. This circular TAR cloning system is highly efficient for the specific isolation of human DNAs from monochromosomal/hybrid cell lines and it can be used to rapidly isolate human DNAs from radiation hybrids containing only a small fragment of a human chromosome. The circular YACs greatly facilitate subsequent physical isolation and analysis of the cloned material.
Additionally, we have expanded this new TAR cloning method to a yeast cloning system that can specifically clone a selected nucleic acid from a mixed population of nucleic acids by incorporating into the cloning vector a sequence or region that is specific for the selected nucleic acid and which recombines with the selected nucleic acid. This specificity of the sequence which recombines with the nucleic acid allows one to specifically target a nucleic acid or nucleic acid species, a cDNA for example, from a mixed population of nucleic acids and specifically clone, and therefore isolate, that individually selected nucleic acid. This allows one to selectively clone a nucleic acid, such as a cDNA, where only part of the sequence of that nucleic acid was previously known. There are an increasing number of cDNAs being generated where only the 3′-ends of these cDNAs are being sequenced. This technique will allow for the selective cloning of these cDNAs based on that information. The remaining sequence information from these nucleic acids can, therefore, be rapidly identified. This complete sequence information will accelerate the determination of the role of these molecules in the organism, and allow investigators to more efficiently and thoroughly determine the roles and interactions of specific genes in the life cycle of that organism. Further, the invention also provides for the expansion of TAR cloning to
E. coli.
SUMMARY OF THE INVENTION
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to a method of making a yeast artificial chromosome comprising introducing into yeast cells a population of nucleic acids and a vector, wherein the vector comprises a yeast centromere, a selectable marker, a yeast telomere, and a sequence which can recombine with a region of a nucleic acid within the population of nuclcic acids; whereby in vivo recombination makes the yeast artificial chromosome.
The invention also provides a method of making a yeast artificial chromosome comprising introducing into ycast cells a population of nucleic acids and 1) a first vector comprising a yeast centromere, a yeast telomere, and a first sequence which can recombine with a region of a nucleic acid within the population of nucleic acids and 2) a second vector comprising a yeast telomere and a second sequence which can recombine with a region of the nucleic acid within the population of nucleic acids; and wherein, at least one of the vectors further comprises a selectable marker; whereby in vivo recombination makes the yeast artificial chromosome.
In another aspect, the invention provides a method of making a circular yeast artificial chromosome comprising introducing into yeast cells a population of nucleic acids and a vector, wherein the vector comprises a yeast centromere, a select
Kouprina Natalay Y.
Larionov Vladimir L.
Perkins Edward L.
Resnick Michael A.
Needle & Rosenberg P.C.
The United States of America as represented by the Department of
Yucel Remy
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