Nucleic acid analysis techniques

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.

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.    
     
     
         2 - 49 . (canceled)  
     
     
         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.  
     
     
         51 . The method according to  claim 50 , wherein said detectably labeled nucleic acid is directly detectable.  
     
     
         52 . The method according to  claim 51 , wherein said directly detectable label is fluorescent.  
     
     
         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.  
     
     
         54 . The method according to  claim 53 , wherein said nucleic acid sample is an mRNA sample.  
     
     
         55 . The method according to  claim 54 , wherein a primer that comprises an oligo dT domain and said oligonucleotide tag is employed in said enzymatically generating step.  
     
     
         56 . The method according to  claim 54 , wherein said primer further comprises an RNA polymerase promoter domain.  
     
     
         57 . The method according to  claim 54 , wherein said population of detectably labeled target nucleic acids is labeled with a fluorescent label.  
     
     
         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.  
     
     
         59 . The method according to  claim 58 , wherein said probe is stably associated with the surface of a solid support.  
     
     
         60 . The method according to  claim 58 , wherein said probe is present on an array.  
     
     
         61 . The method according to  claim 58 , wherein said labeled oligonucleotide is fluorescently labeled.  
     
     
         62 . The method according to  claim 58 , wherein a primer comprising an oligo dT region and said oligonucleotide tag is employed in said enzymatically generating step.  
     
     
         63 . The method according to  claim 62 , wherein said primer further comprises an RNA polymerase promoter.  
     
     
         64 . The method according to  claim 58 , wherein said method further comprises transmitting data obtained from at least one of said detecting and related steps to a remote location.  
     
     
         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.  
     
     
         66 . The method according to  claim 65 , wherein a primer comprising an oligo dT domain and said oligonucleotide tag is employed in said enzymatically generating step.  
     
     
         67 . The method according to  claim 66 , wherein said primer further comprises an RNA polymerase promoter.  
     
     
         68 . The method according to  claim 65 , wherein said labeled oligonucleotide is fluorescently labeled.  
     
     
         69 . The method according to  claim 65 , wherein said method further comprises transmitting data obtained from at least one of said detecting and deriving steps to a remote location.  
     
     
         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.  
     
     
         71 . The method according to  claim 70 , wherein said first and second labels are distinguishable.  
     
     
         72 . The method according to  claim 70 , wherein said first and second labels are fluorescent.  
     
     
         73 . The method according to  claim 70 , wherein a primer comprising an oligo dT domain and said oligonucleotide tag is employed in said enzymatically generating step.  
     
     
         74 . The method according to  claim 73 , wherein said primer further comprises an RNA polymerase promoter.  
     
     
         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.  
     
     
         76 . The kit according to  claim 75 , wherein said primer further comprises an RNA polymerase promoter.  
     
     
         77 . The kit according to  claim 75 , wherein said hybridization domain is an oligo dT domain.  
     
     
         78 . The kit according to  claim 75 , wherein said hybridization domain is a domain of random sequence.  
     
     
         79 . The kit according to  claim 75 , wherein said kit further comprises an array of probe nucleic acids stably associated with the surface of a solid support.  
     
     
         80 . The kit according to  claim 75 , wherein said kit further comprises: (a) a second primer comprising an hybridization domain and a second oligonucleotide tag having a sequence different from said first oligonucleotide tag; and (b) a second labeled oligonucleotide, wherein the label of said second labeled oligonucleotide is distinguishable from the label of said first labeled oligonucleotide.  
     
     
         81 . The kit according to  claim 78 , wherein said second primer further comprises an RNA polymerase promoter.  
     
     
         82 . The kit according to  claim 75 , wherein said kit further comprises an RNA polymerase.  
     
     
         83 . The kit according to  claim 75 , wherein said first labeled oligonucleotide is labeled with a fluorescent label.  
     
     
         84 . The kit according to  claim 75 , wherein said kit further comprises a computer readable storage medium on which is recorded an algorithm for designing oligonucleotide tag sequences.