Use of representations of DNA for genetic analysis

• US 8,663,917 B2
• Filed: 03/30/2005
• Issued: 03/04/2014
• Est. Priority Date: 10/30/1997
• Status: Expired due to Fees

• Current Assignee: Cold Spring Harbor Laboratory
• Original Assignee: Cold Spring Harbor Laboratory
• Inventors: Wigler, Michael, Lucito, Robert
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US11/094,565
• Publication Number:
  US 20050266444A1
• Time in Patent Office: 3,261 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2533/101 Primer extension

Abstract

It is an object of the present invention to provide a solution to problems associated with the use of microarray technology for the analysis DNA. The present invention provides compositions and methods for the use of simple and compound representations of DNA in microarray technology. The present invention is also directed to methods for the production of High Complexity Representations (HCRs) of the DNA from cells.

Claims

1. A process for obtaining genomic copy number information about genomic DNA from a diseased cell of an organism, which does not involve Representational Difference Analysis (RDA), comprising

a) providing a representation of the genomic DNA by digesting the genomic DNA from the diseased cell with a single restriction endonuclease to provide digested DNA fragments, ligating an adaptor to the digested DNA fragments to form ligated DNA fragments, and polymerase chain reaction (PCR) amplifying the ligated DNA fragments using a single set of primers complementary to the adaptor to provide the representation of the genomic DNA;

b) contacting nucleic acids from the representation provided in part (a), and a second representation obtained from genomic DNA from a normal cell from the same organism, with a microarray comprising

i) a solid support with addresses of defined coordinates thereon; and

ii) 1000 predetermined nucleic acid probes, wherein each probe has a different nucleic acid sequence;

is affixed to an address of defined coordinates on the solid support;

has 20 consecutive nucleotides in a sequence which is contained in the sequence of a member of said representation of part (a); and

is mapped to a region of a genome,

under conditions that allow hybridization between the nucleic acids and the nucleic acid probes to occur;

c) detecting the hybridization at addresses of defined coordinates of the microarray in step b),

d) obtaining genomic copy number information based on the hybridization detected in step c).

2. The process of claim 1, wherein the genomic DNA is from a biopsy specimen, a cell line, an autopsy specimen, a forensic specimen or a paleontological specimen.

3. The process of claim 1, wherein the diseased cell is a tumor cell of an organism.

4. The process of claim 1, wherein the genomic DNA is from a non-tumor cell of an organism.

5. The process of claim 3, wherein the normal cell is a non-tumor cell from the same organism.

6. The process of claim 1, wherein the representation of step a) is fluorescently labeled.

7. The process of claim 1, wherein the microarray comprises 10,000 predetermined nucleic acid probes.

8. The process of claim 1, wherein the microarray comprises nucleic acid probes of 100-1000 nucleotides in length.

9. The process of claim 1, wherein the microarray comprises nucleic acid probes fixed to the surface of the solid support at their 5′

ends.

10. The process of claim 1, wherein the microarray further comprises multiple copies of a probe, and wherein multiple copies of the same probe are affixed to an address.

11. The process of claim 1, wherein the microarray comprises a mixture of different nucleic acid probes affixed to an address.

12. The process of claim 1, wherein the probes at each address together have a total nucleotide complexity of less than or equal to 1.2 kb.

13. The process of claim 1, wherein the probes at each address together have a total nucleotide complexity that exceeds 1.2 kb.

14. The process of claim 1, wherein the representation of the genome has a complexity (R %) of 70%≧

R %>

0% of the complexity of the genome.

15. The process of claim 1, wherein the representation of the genome has a complexity (R %) of about 2%≧

R %>

0% of the complexity of the genome.