Methods for analysis of gene expression

Abstract

This invention provides methods, compositions and kits for gene expression analysis and gene expression profiling. The methods of the invention are highly sensitive; have a wide dynamic range; are rapid and inexpensive; have a high throughput; and allow the simultaneous differential analysis of a defined set of genes. The methods, compositions and kits of the invention also provide tools for gene expression data collection and relational data analysis.

1 .- 107 . (canceled) 
     
     
         108 . A method of determining a gene expression profile, comprising:
 a) providing one or more nucleic acid samples;   b) amplifying a plurality of target sequences from the one or more nucleic acid samples thereby producing a set of amplification products, wherein amplifying the plurality of target sequences comprises combining the plurality of target sequences, one or more target-specific primers, and one or more universal primers to form a reaction mixture, and wherein the reaction mixture comprises one or more of i) an excess concentration of the one or more universal primers relative to a target-specific primer, ii) universal primers which have a higher annealing temperature relative to the annealing temperature of a target-specific primer, or iii) modified target-specific primers for attenuation of abundant target genes;   c) providing a solid support comprising a set of nucleic acid members corresponding to a plurality of gene expression products, wherein at least one member of the set is complementary to at least a portion of a member of the set of amplification products;   d) hybridizing member amplification products to complementary member nucleic acids on the solid support; and,   e) detecting hybridized member amplification products on the solid support, thereby determining a gene expression profile.   
     
     
         109 . The method of  claim 108 , wherein a universal primer: target-specific primer concentration ratio ranges from about 5:1 to about 100:1. 
     
     
         110 . The method of  claim 108 , wherein the modified target-specific primers comprise a blocking group attached at a 3′ end of the modified target-specific primer, one or more abasic nucleotides or mismatch nucleotides, a blocking group attached at a 3′ end of a reverse target-specific primer, or a phosphate group on the terminal 3′-hydroxyl of the target-specific primer. 
     
     
         111 . The method of  claim 108 , wherein said plurality of target sequences further comprises one or more reference sequences, wherein a portion of the one or more reference sequences is homologous to at least one member of the plurality of target-specific primers. 
     
     
         112 . The method of  claim 108 , wherein at least one member of the plurality of target-specific primers or universal primers further comprises a modified nucleotide. 
     
     
         113 . The method of  claim 112 , wherein the modified nucleotide prevents amplification of one or more portions of the at least one member of the plurality of target-specific primers or universal primers. 
     
     
         114 . The method of  claim 112 , wherein the modified nucleotide comprises one or more non-nucleotide linkers, alkyl chains, or abasic nucleotides. 
     
     
         115 . The method of  claim 108 , wherein at least one member of the plurality of target-specific primers or universal primers further comprises a cleavable linker. 
     
     
         116 . The method of  claim 108 , wherein at least one universal primer further comprises a label. 
     
     
         117 . The method of  claim 108 , wherein performing amplifying comprises performing a polymerase chain reaction, a transcription-based amplification, a self-sustained sequence replication, a nucleic acid sequence based amplification, a ligase chain reaction, a ligase detection reaction, a strand displacement amplification, a repair chain reaction, a cyclic probe reaction, a rapid amplification of cDNA ends, an invader assay, a solid phase assay, a solution phase assay, or a combination thereof. 
     
     
         118 . The method of  claim 108 , wherein the amplifying and detecting steps are performed at a rate of about 100 samples per hour to about 5,000 samples per hour. 
     
     
         119 . The method of  claim 108 , wherein the solid support comprises a microarray. 
     
     
         120 . A method of determining a gene expression profile, comprising:
 a.) providing one or more nucleic acid samples;   b.) amplifying a plurality of target sequences from the one or more nucleic acid samples thereby producing a set of amplification products, wherein amplifying the plurality of target sequences comprises combining the plurality of target sequences, one or more target-specific primers, and one or more universal primers to form a reaction mixture, wherein the universal primers comprise an attachment moiety, and wherein the reaction mixture comprises one or more of i) an excess concentration of the one or more universal primers relative to a target-specific primer, ii) universal primers which have a higher annealing temperature relative to the annealing temperature of a target-specific primer, or iii) modified target-specific primers for attenuation of abundant target genes;   c.) using the attachment moiety to attach the amplification products to a solid support, and,   d.) detecting the amplification products on the solid support, thereby determining a gene expression profile.   
     
     
         121 . The method of  claim 120 , wherein the attachment moiety comprises biotin.