Colorimetric in situ hybridization detection methods

• US 20030044822A1
• Filed: 04/26/2002
• Published: 03/06/2003
• Est. Priority Date: 11/02/1998
• Status: Abandoned Application

• Current Assignee: Brigham and Women's Hospital Incorporated
• Original Assignee: Brigham and Women's Hospital Incorporated
• Inventors: Fletcher, Jonathan, Xiao, Sheng
• Application Number:
  US10/132,993
• Publication Number:
  US 20030044822A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/137 Chromatographic separation
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The invention is a method for genomic subtractive hybridization. Specific nucleic acid sequences are removed from a sample of nucleic acid sequences by specifically hybridizing the sequences to a complementary nucleic acid sequence bound to a target molecule such as biotin. The target molecule is then contacted with a binding partner such as avidin and separated from the sample of nucleic acid sequences. As the target is separated from the sample the hybridized nucleic acid sequences are also removed from the sample. The method preferably involves the removal of repetitive nucleic acid sequences from a nucleic acid sample to generate a library of probes that are substantially free of repetitive nucleic acid sequences.

Claims

1. A method for genomic subtractive hybridization, comprising:

hybridizing a chemically modified oligonucleotide probe to a complementary nucleic acid sequence in a nucleic acid sample, wherein the chemically modified oligonucleotide probe is an oligonucleotide associated with a target molecule, and

selectively removing the chemically modified oligonucleotide probe and complementary nucleic acid by selectively contacting the target molecule with a binding partner to produce binding partner/target conjugates and separating the binding partner and binding partner/target conjugates from the nucleic acid sample.

2. The method of claim 1, wherein the target molecule is selected from the group consisting of avidin, biotin, FITC, anti-FITC, antigen, and antibodies.

3. The method of claim 1, wherein the oligonucleotide is a repetitive nucleic acid.

4. The method of claim 3, wherein the repetitive nucleic acid is isolated from a DNA source selected from the group consisting of YAC, BAC and PAC DNA.

5. The method of claim 3, wherein the chemically modified oligonucleotide probe is prepared by amplifying the repetitive nucleic acid using PCR and a primer attached to the target molecule.

6. The method of claim 1, wherein the nucleic acid sample is a genomic nucleic acid sample.

7. The method of claim 1, wherein the genomic nucleic acid sample is obtained from a YAC clone.

8. The method of claim 7, wherein the YAC clone is purified by pulsed field gel electrophoresis.

9. The method of claim 1, wherein the binding partner is immobilized on a support.

10. The method of claim 9, wherein the support is a bead and wherein the binding partner/target conjugates are separated from the nucleic acid sample by chromatography.

11. The method of claim 1, wherein the genomic nucleic acid sample is obtained from a DNA source selected from the group consisting of YAC, BAC and PAC DNA.

12. A library of nucleic acid probes for use in in situ hybridization methods, comprising:

a heterogenous mixture of labeled nucleic acid probes that are substantially complementary to unique nucleic acid fragments and are substantially free of repetitive nucleic acid sequences, and which are produced by the process of;

(a) obtaining genomic nucleic acid fragments;

(b) amplifying the genomic nucleic acid fragments;

(c) hybridizing a chemically modified oligonucleotide probe to a complementary nucleic acid sequence in the genomic nucleic acid fragments, wherein the chemically modified oligonucleotide probe is an oligonucleotide associated with a target molecule;

(d) selectively removing the chemically modified oligonucleotide probe and complementary nucleic acid by selectively contacting the target molecule with a binding partner and separating the binding partner and binding partner/target conjugates from the genomic nucleic acid fragments; and

(e) labeling the genomic nucleic acid fragments with a label.

13. The library of claim 12, wherein the genomic nucleic acid fragments are labeled with a fluorescent molecule.

14. The library of claim 12, wherein the heterogenous mixture of labeled nucleic acid probes are complementary to substantially all of a genomic DNA population.

15. The library of claim 12, wherein the heterogenous mixture of labeled nucleic acid probes are complementary to substantially all of a chromosome of a genomic DNA population.

16. The library of claim 12, wherein the heterogenous mixture of labeled nucleic acid probes are complementary to substantially all of a subregion of a chromosome of a genomic DNA population.

17. A method for performing in situ hybridization, comprising:

hybridizing the library of labeled probes of claim 12 to a biological sample fixed on a surface,

removing un-hybridized probe, and

detecting a signal from the hybridized probe to identify nucleic acid sequences present in the biological sample.