Use of representations of DNA for genetic analysis

• US 7,531,307 B2
• Filed: 03/30/2005
• Issued: 05/12/2009
• Est. Priority Date: 10/30/1997
• Status: Expired due to Fees

• Current Assignee: Cold Spring Harbor Laboratory
• Original Assignee: Cold Spring Harbor Laboratory
• Inventors: Lucito, Robert, Wigler, Michael
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): CALAMITA, HEATHER
• Application Number:
  US11/094,388
• Publication Number:
  US 20050196799A1
• Time in Patent Office: 1,504 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 method of producing a compound representation of DNA, comprising:

(a) digesting a sample of DNA with a first restriction endonuclease to provide digested DNA fragments;

(b) ligating an adaptor to said digested DNA fragments;

(c) amplifying DNA fragments created in step b) using primers complementary to said adaptor to provide a first representation of said DNA;

(d) digesting the first representation with a second restriction endonuclease to provide digested DNA fragments, wherein said second restriction endonuclease is different from said first restriction endonuclease; and

(e) amplifying said DNA fragments of step (d) using primers which are the same as the primers used in step c) to provide a compound representation of said DNA.

4. The method according to claim 1, wherein said DNA is obtained from a biopsy specimen, a cell line, an autopsy specimen, a forensic specimen or a paleontological specimen.

5. The method according to claim 1, wherein said DNA is obtained from a fractionated cell or tissue sample.

12. A method of producing a microarray comprising

(a) producing a compound representation of DNA by the method of claim 1;

and

(b) attaching to a solid support, probes having a nucleotide sequence corresponding to DNA fragments in the compound representation, thereby producing the microarray.

2. A method of producing a compound representation of DNA, comprising:

(a) digesting a sample of DNA with a first restriction endonuclease to provide digested DNA fragments;

(b) ligating a first adaptor to said digested DNA fragments;

(c) amplifying DNA fragments created in step b) using primers complementary to said first adaptor to provide a first representation of said DNA;

(d) digesting the first representation with a second restriction endonuclease to provide digested DNA fragments, wherein said second restriction endonuclease is different from said first restriction endonuclease;

(e) ligating a second adaptor to the ends of said digested DNA fragments created by the second restriction endonuclease in step d);

(f) ligating a third adaptor to the 5′

ends of DNA fragments create in step c) and not cleaved by the second restriction endonuclease in step (d); and

(g) amplifying said DNA fragments of step (e) and (f) using primers complementary to the second and third adaptors to provide a compound representation of said DNA.

6. The method according to claim 2, wherein said DNA is obtained from a biopsy specimen, a cell line, an autopsy specimen, a forensic specimen or a paleontological specimen.

7. The method according to claim 2, wherein said DNA is obtained from a fractionated cell or tissue sample.

13. A method of producing a microarray comprising

(a) producing a compound representation of DNA by the method of claim 2;

and

(b) attaching to a solid support, probes having a nucleotide sequence corresponding to DNA fragments in the compound representation, thereby producing the microarray.

3. A method of producing a compound representation of DNA, comprising:

(a) digesting a sample of DNA with a first restriction endonuclease to provide digested DNA fragments;

(b) ligating an adaptor to said digested DNA fragments;

(c) digesting DNA fragments created in step b) with a second restriction endonuclease to provide double-digested DNA fragments, wherein said second restriction endonuclease is different from said first restriction endonuclease; and

(d) amplifying said double-digested DNA fragments using primers complementary to said adaptor to provide a compound representation of said DNA.

8. The method according to claim 3, wherein said DNA is obtained from a biopsy specimen, a cell line, an autopsy specimen, a forensic specimen or a paleontological specimen.

9. The method according to claim 3, wherein said DNA is obtained from a fractionated cell or tissue sample.

14. A method of producing a microarray comprising

(a) producing a compound representation of DNA by the method of claim 3;

and

(b) attaching to a solid support, probes having a nucleotide sequence corresponding to DNA fragments in the compound representation, thereby producing the microarray.

10. A method of producing a high complexity representation of DNA, comprising:

(a) digesting a sample of DNA with at least two restriction endonucleases to provide digested DNA fragments in which at least about 20% of said fragments are at between 200 and 1200 bp, wherein the second restriction endonuclease is different from the first restriction endonuclease;

(b) ligating adaptors to said digested DNA fragments; and

(c) amplifying DNA fragments created in step b) using primers complementary to said adaptors to provide a high complexity representation of said DNA.

11. The method of claim 10 in which the DNA sample is a genomic DNA sample and the high complexity representation represents about 20-90% of the genome.

15. A method of producing a microarray comprising

(a) producing a high complexity representation of DNA by the method of claim 10;

and

(b) attaching to a solid support, probes having a nucleotide sequence corresponding to DNA fragments in the compound representation, thereby producing the microarray.