Systems and Methods to Quantify and Amplify Both Signaling and Probes for CDNA Chips and Gene Expression Microarrays

Abstract

The invention provides a series of reagent compositions and methods for making and amplifying novel cDNA based probe sets from RNA samples to improve analysis with gene expression arrays. The methods globally produce probe sets with common universal linkers at one or both ends, called WRAP-Probes, wherein the linkers do not bind to the target sequences and they can efficiently bind added reporters to the probes. The universal linkers are also designed as primer binding sites for copying and amplifying the probes, either linearly with one linker, or exponentially with double linkers. The capacity to globally and exponentially amplify the probe set by PCR is a primary advantage. Adding reporters by terminal linkers also improves quantification since each probe gets equivalent signaling. The invention allows expression analysis of small research, clinical and forensic samples to enable improved diagnostics, drug discovery, therapeutic monitoring, and medical, agricultural and general research.

1 .- 49 . (canceled) 
     
     
         50 . A method of gene expression analysis, the method comprising:
 (a) providing mRNA transcripts from a sample;   (b) making cDNA probes from the mRNA transcripts, wherein the cDNA probes comprise a universal linker;   (c) hybridizing the cDNA probes to an array;   (d) providing labeled reporter molecules, wherein the reporter molecules bind to the universal linkers of the probes; and   (e) detecting the labeled reporter molecules to determine gene expression in the sample.   
     
     
         51 . The method of  claim 50 , wherein the cDNA probe comprises one universal linker. 
     
     
         52 . The method of  claim 51 , wherein the step of making the cDNA probes comprise using a modified poly-T primer comprising a universal linker at the 5′-end of the primer. 
     
     
         53 . The method of  claim 52 , wherein the modified poly-T-primer comprises a poly-thymidine sequence at a 3′-end of the primer, a universal linker at the 5′-end of the primer, wherein the poly-thymidine sequence comprises about 12 to about 20 thymidine bases. 
     
     
         54 . The method of  claim 53 , wherein the modified poly-T primer further comprises an anchor sequence at the 3′-end of the primer. 
     
     
         55 . The method of  claim 54 , wherein the anchor sequence comprises the sequence 5′-poly-T, V, N-3′, wherein V is a variable base selected from the group consisting of adenine, guanine, or cytosine, and wherein N is randomly any base selected from the group consisting of adenine, guanine, cytosine, or thymidine. 
     
     
         56 . The method of  claim 53 , wherein the modified poly-T primer comprises a capture moiety. 
     
     
         57 . The method of  claim 56 , wherein the capture moiety is biotin. 
     
     
         58 . The method of  claim 50 , wherein the universal linker comprises the sequence of SEQ ID NO:1, 2, 3, or 4. 
     
     
         59 . The method of  claim 50 , wherein the cDNA probes comprise two universal linkers. 
     
     
         60 . The method of  claim 59 , wherein the step of making the cDNA probes comprise using a modified poly-T primer comprising a universal linker at the 5′-end of the primer. 
     
     
         61 . The method of  claim 60 , wherein the modified poly-T-primer comprises a poly-thymidine sequence at a 3′-end of the primer, a universal linker at the 5′-end of the primer, wherein the poly-thymidine sequence comprises about 12 to about 20 thymidine bases. 
     
     
         62 . The method of  claim 61 , wherein the modified poly-T primer further comprises an anchor sequence at the 3′-end of the primer. 
     
     
         63 . The method of  claim 62 , wherein the anchor sequence comprises the sequence 5′-poly-T, V, N-3′, wherein V is a variable base selected from the group consisting of adenine, guanine, or cytosine, and wherein N is randomly any base selected from the group consisting of adenine, guanine, cytosine, or thymidine. 
     
     
         64 . The method of  claim 61 , wherein the modified poly-T primer comprises a capture moiety. 
     
     
         65 . The method of  claim 64 , wherein the capture moiety is biotin. 
     
     
         66 . The method of  claim 59 , wherein the two universal linker sequences comprise the sequence of SEQ ID NO:1, 2, 3, or 4. 
     
     
         67 . The method of  claim 66 , wherein the two universal linker sequences comprise the same sequence or different sequences. 
     
     
         68 . A probe set comprising a pool of cDNA probes, wherein each probe comprises a universal linker sequence at one or both ends of the probe, and wherein the universal linker sequence comprises the sequence of SEQ ID NO:1, 2, 3, or 4. 
     
     
         69 . A modified poly-T primer composition comprising a poly-thymidine sequence at the 3′-end of the primer and a universal linker at the 5′-end of the primer, wherein the universal linker comprises the sequence of SEQ ID NO:1, 2, 3, or 4. 
     
     
         70 . The modified poly T-primer of  claim 69 , wherein the poly-thymidine sequence comprises about 12 to about 20 thymidine bases. 
     
     
         71 . The modified poly T-primer of  claim 70 , further comprising an anchor sequence at the 3′-end of the primer. 
     
     
         72 . The modified poly T-primer of  claim 71 , wherein the anchor sequence comprises the sequence 5′-poly-T, V, N-3′, wherein V is a variable base selected from the group consisting of adenine, guanine, or cytosine, and wherein N is randomly any base selected from the group consisting of adenine, guanine, cytosine, or thymidine. 
     
     
         73 . The modified poly T-primer of  claim 70 , further comprising a capture moiety. 
     
     
         74 . The modified poly T-primer of  claim 73 , wherein the capture moiety is biotin. 
     
     
         75 . An adapter composition for providing a second universal linker to a cDNA probe set, the composition comprising a first and second polynucleotide sequence, wherein the first polynucleotide sequence is a universal linker sequence comprising the sequence of SEQ ID NO:1, 2, 3, or 4 and the second polynucleotide sequence is complimentary to the first polynucleotide sequence, and wherein the first or second polynucleotide sequence comprises an additional single-strand overhang. 
     
     
         76 . The adapter composition of  claim 75 , wherein the single-strand overhang comprises about 1 to about 6 nucleotides, and wherein the single-strand overhang is complimentary to a specific restriction enzyme cut site. 
     
     
         77 . The adapter composition of  claim 75 , wherein the single-strand overhang comprises about 1 to about 6 nucleotides, and wherein the nucleotides are randomly and alternatively selected from the group consisting of an adenine (A), a thymidine (T), a cytosine (C), and a guanine (G). 
     
     
         78 . The adapter composition of  claim 75 , wherein single-strand overhang comprises a poly-cytosine (poly-C) or a poly-guanine (poly-G) sequence. 
     
     
         79 . An extender composition for providing a second universal linker to a cDNA probe set, the composition comprising a single-stranded polynucleotide sequence comprising a universal linker sequence at the 5′-end, wherein the universal linker sequence comprises the sequence of SEQ ID NO:1, 2, 3, or 4. 
     
     
         80 . The extender composition of  claim 79 , wherein the polynucleotide sequence comprises a random sequence at the 3′-end, wherein the random sequence comprises about 4 to about 10 nucleotides. 
     
     
         81 . The extender composition of  claim 80 , wherein the 3′-end comprises a block to prevent polymerase extension. 
     
     
         82 . The extender composition of  claim 79 , wherein the polynucleotide sequence comprises a poly-C or a poly-G sequence, wherein the poly-C or poly-G sequences comprise about 5 to about 15 nucleotides.