Nucleic acid analysis techniques
First Claim
1. A method of identifying differences in nucleic acid levels between two or more nucleic acid samples, said method comprising the steps of:
- (a) providing one or more oligonucleotide arrays said arrays comprising probe oligonucleotides attached to a surface;
(b) hybridizing said nucleic acid samples to said one or more arrays to form hybrid duplexes between nucleic acids in said nucleic acid samples and probe oligonucleotides in said one or more arrays that are complementary to said nucleic acids or subsequences thereof;
(c) contacting said one or more arrays with a nucleic acid ligase; and
(d) determining differences in hybridization between said nucleic acid samples wherein said differences in hybridization indicate differences in said nucleic acid levels.
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Abstract
The present invention provides a simplified method for identifying differences in nucleic acid abundances (e.g., expression levels) between two or more samples. The methods involve providing an array containing a large number (e.g. greater than 1,000) of arbitrarily selected different oligonucleotide probes where the sequence and location of each different probe is known. Nucleic acid samples (e.g. mRNA) from two or more samples are hybridized to the probe arrays and the pattern of hybridization is detected. Differences in the hybridization patterns between the samples indicates differences in expression of various genes between those samples. This invention also provides a method of end-labeling a nucleic acid. In one embodiment, the method involves providing a nucleic acid, providing a labeled oligonucleotide and then enzymatically ligating the oligonucleotide to the nucleic acid. Thus, for example, where the nucleic acid is an RNA, a labeled oligoribonucleotide can be ligated using an RNA ligase. In another embodiment, the end labeling can be accomplished by providing a nucleic acid, providing labeled nucleoside triphosphates, and attaching the nucleoside triphosphates to the nucleic acid using a terminal transferase.
107 Citations
84 Claims
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1. A method of identifying differences in nucleic acid levels between two or more nucleic acid samples, said method comprising the steps of:
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(a) providing one or more oligonucleotide arrays said arrays comprising probe oligonucleotides attached to a surface;
(b) hybridizing said nucleic acid samples to said one or more arrays to form hybrid duplexes between nucleic acids in said nucleic acid samples and probe oligonucleotides in said one or more arrays that are complementary to said nucleic acids or subsequences thereof;
(c) contacting said one or more arrays with a nucleic acid ligase; and
(d) determining differences in hybridization between said nucleic acid samples wherein said differences in hybridization indicate differences in said nucleic acid levels.
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2-49. -49. (canceled)
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50. A method for producing a detectably labeled nucleic acid, said method comprising:
- enzymatically generating by template dependent polymerization an oligonucleotide tagged nucleic acid, wherein said oligonucleotide tagged nucleic acid comprises an oligonucleotide tag; and
contacting said oligonucleotide tagged nucleic acid with a labeled oligonucleotide complementary to said oligonucleotide tag under conditions sufficient for said labeled oligonucleotide to hybridize to said oligonucleotide tag;
whereby a detectably labeled nucleic acid is produced. - View Dependent Claims (51, 52)
- enzymatically generating by template dependent polymerization an oligonucleotide tagged nucleic acid, wherein said oligonucleotide tagged nucleic acid comprises an oligonucleotide tag; and
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53. A method for producing a detectably labeled population of target nucleic acids from an initial nucleic acid sample, said method comprising:
- enzymatically generating a population of oligonucleotide tagged target nucleic acids from an initial nucleic acid sample, wherein said oligonucleotide tagged target nucleic acids in said population comprise the same oligonucleotide tag; and
contacting said population of oligonucleotide tagged target nucleic acids with labeled oligonucleotides complementary to said oligonucleotide tag of each oligonucleotide tagged target nucleic acid under hybridization conditions;
whereby a detectably labeled population of target nucleic acids is produced. - View Dependent Claims (54, 55, 56, 57)
- enzymatically generating a population of oligonucleotide tagged target nucleic acids from an initial nucleic acid sample, wherein said oligonucleotide tagged target nucleic acids in said population comprise the same oligonucleotide tag; and
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58. A method of detecting the presence of a nucleic acid analyte in a target sample, said method comprising:
- (a) enzymatically generating an oligonucleotide tagged target nucleic acid from said nucleic acid analyte, wherein said oligonucleotide tagged target nucleic acid comprises an oligonucleotide tag; and
(b) producing a hybridized complex that comprises;
(i) said tagged target nucleic acid;
(ii) an oligonucleotide label; and
(iii) a probe nucleic acid;
(c) detecting the presence of said hybridized complex; and
(d) relating the presence of said hybridized complex to the presence of said nucleic acid analyte in said sample;
whereby the presence of said nucleic acid analyte in said sample is detected. - View Dependent Claims (59, 60, 61, 62, 63, 64)
- (a) enzymatically generating an oligonucleotide tagged target nucleic acid from said nucleic acid analyte, wherein said oligonucleotide tagged target nucleic acid comprises an oligonucleotide tag; and
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65. A method for obtaining an expression profile for at least a representative number of genes in a cell, said method comprising:
- (a) enzymatically generating a population of oligonucleotide tagged nucleic acids from an mRNA sample derived from said cell, wherein each oligonucleotide tagged nucleic acid comprises an oligonucleotide tag;
(b) producing at least one hybridized complex comprising;
(i) a tagged target nucleic acid;
(ii) a labeled oligonucleotide; and
(iii) a probe nucleic acid stably associated with the surface of a solid support;
(c) detecting the presence of said at least one hybridized complex on said array surface; and
(d) deriving an expression profile for said cell from said detected at least one hybridized complex;
whereby said expression profile for at least a representative number of genes in said cell is obtained. - View Dependent Claims (66, 67, 68, 69)
- (a) enzymatically generating a population of oligonucleotide tagged nucleic acids from an mRNA sample derived from said cell, wherein each oligonucleotide tagged nucleic acid comprises an oligonucleotide tag;
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70. A method for comparing the expression profiles of at least two distinct samples, said method comprising:
- (a) enzymatically generating;
(i) a first population of oligonucleotide tagged nucleic acids from an mRNA sample derived from a first sample, wherein each oligonucleotide tagged nucleic acid of said first population comprises a first oligonucleotide tag; and
(ii) a second population of oligonucleotide tagged nucleic acids from an mRNA sample derived from a second sample, wherein each oligonucleotide tagged nucleic acid of said second population comprises a second oligonucleotide tag that differs from said first oligonucleotide tag;
(b) hybridizing said first and second populations of oligonucleotide tagged target nucleic acids to an array of probe nucleic acids stably associated with the surface of a solid support, wherein said hybridizing occurs in the presence of;
(i) first labeled oligonucleotide complementary to said first oligonucleotide tag of said first population; and
(ii) second labeled oligonucleotide complementary to said second oligonucleotide tag of said second population;
whereby hybridized complexes comprising said oligonucleotide tagged target nucleic acids, probe nucleic acids and labeled oligonucleotides are produced on the surface of said array;
(c) detecting the presence of said hybridized complexes on said array surface;
(d) deriving an expression profile for each of said cells from said detected hybridized complexes; and
(e) comparing the derived expression profiles of each of said cells;
whereby the expression profiles of said at least two distinct cells are compared. - View Dependent Claims (71, 72, 73, 74)
- (a) enzymatically generating;
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75. A kit for use in obtaining an expression profile for at least a representative number of genes in a cell, said kit comprising:
- (a) a first primer comprising an hybridization domain and an oligonucleotide tag; and
(b) a first labeled oligonucleotide complementary to said oligonucleotide tag. - View Dependent Claims (76, 77, 78, 79, 80, 81, 82, 83, 84)
- (a) a first primer comprising an hybridization domain and an oligonucleotide tag; and
Specification