التفاصيل البيبلوغرافية
| العنوان: |
Methods for high throughput genome analysis using restriction site tagged microarrays |
| Document Number: |
20050064406 |
| تاريخ النشر: |
March 24, 2005 |
| Appl. No: |
10/475352 |
| Application Filed: |
April 22, 2002 |
| مستخلص: |
A method for high-throughput analysis of genomic material originating from complex biological systems, including complex microbial systems and a method of detecting changes in a genomic material using restriction site tagged (RST) microarrays and sequence passporting technique (in particular microarrays containing NotI-clones). Using the present invention method, methylation or silencing of specific alleles, homozygous and hemizygous deletions, epigenetic factors, genetic predisposition, etc, information which is particularly useful in diagnosis and treatment of cancer diseases, can be detected. The RST microarrays and passporting can also be used for qualitative and quantitative analysis of complex microbial systems. |
| Inventors: |
Zabarovsky, Eugene (Upplands, SE); Ernberg, Ingemar (Enebyberg, SE); Li, Jingfeng (Solna, SE); Protopopov, Alexei (Brookline, MA, US); Wahlestedt, Claes (Stockholm, SE); Kashuba, Vladimir (Spanga, SE); Zabarovska, Veronika (Upplands Vasby, SE) |
| Claim: |
1. Method for preparing nucleic acid or and/or modified nucleic acid reference material bound to a solid phase, comprising steps of: digesting nucleic acid and/or modified nucleic acid reference material using biochemical and/or chemical approaches, to obtain sequence fragments surrounding a specific restriction enzyme recognition site, selecting said nucleic acid and/or modified nucleic acid sequence fragments flanking a specific restriction enzyme recognition site. |
| Claim: |
2. Method according to claim 1, wherein said reference material is digested by a first restriction enzyme and/or one or more second restriction enzymes. |
| Claim: |
3. Method according to claim 2, wherein the restriction enzymes are endonucleases. |
| Claim: |
4. Method according to claim 3, wherein the recognition sites of the first endonuclease is scarcely distributed along said genomic material and is located adjacent to gene sequences, and the recognition sites of said one or more second restriction endonucleases are more frequently occurring along said genomic material than the sites of the first endonuclease. |
| Claim: |
5. Method of claim 4, wherein the digestion by the first and second restriction endonucleases are performed simultaneously, and different linkers are ligated to the ends resulting from cutting by the first and second restriction endonucleases, respectively, which linkers are designed such that when primers are added in order to make PCR reactions, only the fragments containing ends resulting from cutting by the first restriction endonuclease will be amplified. |
| Claim: |
6. Method of claim 4, wherein the reference material is first digested by the one or more second restriction endonucleases, the ends of the thus obtained fragments are self-ligated into the form of circular nucleic acid and/or modified nucleic acid molecules, and any linear fragments remaining after self-ligation are inactivated before digestion with the first restriction endonuclease, whereby the linear fragments resulting from the digestion by the first endonuclease are subjected to PCR amplification. |
| Claim: |
7. Method of claim 2, wherein the first restriction endonuclease in NotI, or any other restriction endonuclease, the restriction sites of which occurs in proximity to CpG islands in the genomic material. |
| Claim: |
8. Method of claim 2, wherein the first restriction endonuclease is NotI, PmeI or Sbfl, or a combination of two or more of said endonucleases, and the second endonuclease is BamHI, BclI, BglII or Sau3A, or a combination of two or more of said endonucleases. |
| Claim: |
9. Method according to claim 1, wherein said nucleic acid and/or modified nucleic acid reference material is selected from RNA, DAN, peptides or modified oligonucleotides, or a combination of two or more of said materials. |
| Claim: |
10. Method according to claim 1, wherein the solid phase is a glass slide, coded beads, cellulose, such as nitrocellulose, or filters. |
| Claim: |
11. Method of claim 1, wherein the genomic material is derived from one or more humans, from different locations in the body/bodies and at the same or different points in time. |
| Claim: |
12. Method of claim 1, wherein the genomic material is derived from bacteria from the gut, skin or other parts of the human body. |
| Claim: |
13. Method of claim 1, wherein the genomic material is derived from any organism, bacteria, animal, or plant, or product produced there from, or from any substance wherein genomic material can be contained, especially air and water. |
| Claim: |
14. Use of representation of the genome, or of a part thereof, of an organism, comprising multiple copies of the nucleic acid and/or modified nucleic acid fragments, or a selection thereof, obtained by means of the method of claim 1 in discriminating between different genomes, detecting methylations, deletions, mutations and other changes within genomic material obtained from the same individual at different points of time, or in the genomic material obtained from one individual as compared to a standard representation obtained from at least one other individual, or a combination thereof. |
| Claim: |
15. Use of the representation according to claim 14, wherein the representation in liquid form is hybridized to the nucleic acid and/or modified nucleic acid fragments present in the form of said solid phases. |
| Claim: |
16. Use of the representation of the genome, or of a part thereof, of an organism, comprising multiple copies of the nucleic acid and/or modified nucleic acid fragments, or a selection thereof, obtained by means of the method of claim 1 for: studying methylation and copy number changes in eukaryotic genomes for diagnosis, prognosis, identification of cancer causing genes, etc, genotyping different microorganisms (viruses, prokaryotic, eukaryotic), studying biocomplexity and diversity of complex biological systems, i.e. human gut, bacterial flora in water, food, air resources, identifying pathogenic organisms in different sources including complex biological mixtures, producing passports (images of microarrays hybridizations, database containing tag sequences) for different purposes: to describe organisms at different conditions, i.e. different ages, disease/healthy, infected/uninfected etc, identifying new organisms, e.g. bacterial species, producing microarrays (DNA-and oligo-based) to study all above described features, verification and maintenance of large biological collections/banks, i.e. verifying cell lines and individual organisms for higher organisms and confirming the purity of the particular strain for microbial species, producing kits for labeling and hybridization with microarrays, producing kits for making sequence tagging (passporting), and producing oligo microarrays to analyze sequence tags. |
| Claim: |
17. Use of the representation according to claim 16, wherein the representation in liquid form is hybridized to the nucleic acid and/or modified nucleic acid fragments present in the form of said solid phases. |
| Claim: |
18. NotI genomic subtraction method for cloning deleted sequences (CODE-genomic subtraction method) based on the use of fragments obtained by the method for preparing nucleic acid or and/or modified nucleic acid reference material bound to a solid phase, comprising the steps of digesting nucleic acid and/or modified nucleic acid reference material using biochemical and/or chemical approaches, to obtain sequence fragments surrounding a specific restriction enzyme recognition site, selecting said nucleic acid and/or modified nucleic acid sequence fragments flanking a specific restriction enzyme recognition site. |
| Claim: |
19-20. (canceled) |
| Current U.S. Class: |
435006/000 |
| رقم الانضمام: |
edspap.20050064406 |
| قاعدة البيانات: |
USPTO Patent Applications |
