Claims
- 1. A method for detecting genetic aberrations, the method comprising: a) using a protocol selected for either performing comparative genomic hybridization (CGH) on a nucleic acid array, or performing gene-expression analysis using RNA; b) performing CGH, or optionally performing gene-expression, respectively.
- 2. The method according to claim 1, wherein the protocol for performing CGH comprises the steps of:
a) reducing autofluorescence on a substrate containing an array of oligonucleotides; the autofluorescence reducing step further comprising:
(1) providing a substrate having a first surface with a functional group for binding unmodified oligonucleotides; (2) arraying a set of target oligonucleotides onto said first surface; (3) treating at least a portion of the first surface with a reducing agent; b) applying an amount of genomic DNA (gDNA) probes, without amplifying said gDNA, of about 10 μg or less; c) labeling gDNA from a test sample and a reference sample with a first fluorescent dye and a second fluorescent dye, respectively; d) pretreating said first surface of said substrate with a blocking reagent to reduce non-specific binding of said gDNA probes to said first surface or target oligonucleotides; e) increasing local concentration of gDNA probes to promote hybridization efficiency and optimizing stringency to promote specificity by means of a predetermined hybridization mixture; f) hybridizing said gDNA probes to said target oligonucleotides; g) treating again, during hybridization, said first surface of said substrate with said blocking reagent to further minimize non-specific binding of gDNA probes to said target oligonucleotides or the first surface; h) imaging the relative fluorescence intensity of said first and second fluorescent dyes.
- 3. The method according to claim 1, wherein the protocol for performing gene-expression comprises the steps of:
a) reducing autofluorescence on a substrate containing an array of biomolecules;
the autofluorescence reducing step further comprising:
(1) providing a substrate having a first surface with a functional group for binding of an unmodified biomolecule; (2) arraying a set of target biomolecules onto said first surface; (3) treating at least a portion of the first surface with a reducing agent; b) applying an amount of either total RNA or mRNA, without amplification, of about 10 μg or less. c) labeling by fluorescent means a number of cDNA probes, which are generated by reverse transcription from either said total RNA or mRNA using either random primers, semi-random primers, anchored dT, or a combination thereof; d) pretreating said first surface of said substrate with a blocking reagent to reduce non-specific binding of said cDNA probes to said first surface or target biomolecules; e) increasing local concentration of cDNA probes to promote hybridization efficiency and optimizing stringency to promote specificity by means of a predetermined hybridization mixture; f) hybridizing a pool of complementary cDNA probes to the target biomolecules; g) treating again during hybridization said first surface of said substrate with said blocking reagent to reduce non-specific binding of said cDNA probes to said first surface or target biomolecules; h) imaging said first surface to determine the relative fluorescence ratio of hybridized cDNA probes and target biomolecules. i) analyzing said fluorescence ratio to determine relative gene copy numbers.
- 4. The method according to claim 2, wherein said imaging step determines a ratio of said first and second fluorescent dyes to represent a copy number of a gene.
- 5. A method for performing comparative genomic hybridization (CGH) on a oligonucleotide-based DNA array, the method comprising the steps of:
a) providing a substrate containing an array of oligonucleotides; b) reducing autofluorescence of the oligonucleotides; c) applying an amount of genomic DNA (gDNA) probes, without amplifying said gDNA, of about 10 μg or less; d) labeling gDNA from a test sample and a reference sample with a first fluorescent dye and a second fluorescent dye, respectively; e) pretreating a first surface of said substrate with a blocking reagent to reduce non-specific binding of said gDNA probes to said first surface or target oligonucleotides; f) hybridizing said gDNA probes to said target oligonucleotides; g) imaging the relative fluorescence intensity of said first and second fluorescent dyes to determine a ratio of said first and second fluorescent dyes to represent a copy number of a gene.
- 6. The method according to claim 5, wherein the method further comprises eliminating autofluorescence of said oligonucleotides according to the steps of: providing a substrate having a first surface with a functional group for binding unmodified oligonucleotides; arraying a set of target oligonucleotides onto said first surface; treating at least a portion of the first surface with a reducing agent.
- 7. The method according to claim 5, wherein the method further comprises increasing local concentration of gDNA probes to promote hybridization efficiency and optimizing stringency to promote specificity by means of a predetermined hybridization mixture.
- 8. The method according to claim 5, wherein the method further comprises treating again, during hybridization, said first surface of said substrate with said blocking reagent.
- 9. The method according to claim 5, wherein the amount of gDNA applied, without amplifying said gDNA, is about 1-10 μg.
- 10. The method according to claim 5, wherein the amount of gDNA applied, without amplifying said gDNA, is about 5 μg or less.
- 11. The method according to claim 5, wherein said oligonucleotides have a length of about 15 bases to about 110 bases.
- 12. The method according to claim 5, wherein said oligonucleotides have a length between about 40 bases to about 100 bases.
- 13. The method according to claim 5, wherein said gDNA is not amplified by PCR amplification.
- 14. An oligonucleotide-based microarray for performing genetic-aberration analysis using total genomic DNA without amplifying, according to the method of claim 5.
- 15. A method for performing RNA expression analysis, the method comprising the steps of:
a) reducing autofluorescence on a substrate containing an array of biomolecules; b) applying an amount of either total RNA or mRNA, without amplification, of about 10 μg or less. c) labeling by fluorescent means a number of cDNA probes, which are generated by reverse transcription from either said total RNA or mRNA using either random primers, semi-random primers, anchored dT, or a combination thereof; d) pretreating a first surface of said substrate with a blocking reagent to reduce non-specific binding of said cDNA probes to said target biomolecules or first surface; e) hybridizing a pool of complementary cDNA probes to the target biomolecules; f) analyzing said fluorescence ratio to determine relative differential gene expression levels.
- 16. The method according to claim 15, wherein the method further comprises the autofluorescence reducing step further comprising: providing a substrate having a first surface with a functional group for binding of an unmodified biomolecule; arraying a set of target biomolecules onto said first surface; treating at least a portion of the first surface with a reducing agent.
- 17. The method according to claim 15, wherein the method further comprises increasing local concentration of cDNA probes to promote hybridization efficiency and optimizing stringency to promote specificity by means of a predetermined hybridization mixture.
- 18. The method according to claim 15, wherein the method further comprises treating again, during hybridization, said first surface of said substrate with said blocking reagent.
- 19. The method according to claim 15, wherein the method further comprises imaging said first surface to determine the relative fluorescence ratio of hybridized cDNA probes and target biomolecules.
- 20. The method according to claim 15, wherein said amount of either total RNA or mRNA applied, without amplification, is about 5 μg or less.
- 21. The method according to claim 15, wherein said amount of mRNA applied, without amplification, is about 0.2 μg or less.
- 22. The method according to claim 15, wherein said cDNA probes have a length of between about 100 bases to about 7 kilobases.
- 23. The method according to claim 22, wherein said CDNA probes have a length of between about 400 bases to about 5 kilobases.
- 24. A microarray for performing gene expression using total RNA, mRNA, aRNA, cRNA, or other RNA, without need for amplification, according to the method of claim 15.
CLAIM OF PRIORITY
[0001] The present Application claims benefit of priority from U.S. Provisional Application No. 60/329,590, filed on Oct. 15, 2001, the entire content of which is incorporated herein.
Provisional Applications (1)
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Number |
Date |
Country |
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60329590 |
Oct 2001 |
US |