Proportional amplification of nucleic acids

Abstract

The proportional amplification of nucleic acids can increase the amount of nucleic acids while preserving the relative abundance of the individual nucleic acid species, or portions thereof, in the original sample. A proportionally amplified nucleic acid preparation may be analyzed in a gene expression monitoring system, preferably involving a nucleic acid probe array.

We claim:  
     
         1 . A method for the proportional amplification of nucleic acid, said method comprising: 
 creating fragments of a single-stranded DNA population;    synthesizing double-stranded DNA from said fragments of a single-stranded DNA population; and    producing multiple copies of sense RNA from said double-stranded DNA.    
     
     
         2 . The method of  claim 1 , wherein said single-stranded DNA population is produced from a nucleic acid population selected from the group consisting of one or more of the following: genomic DNA, cDNA, total RNA, poly(A) +  RNA, and oligonucleotides.  
     
     
         3 . The method of  claim 2 , wherein said poly(A) +  RNA is mRNA.  
     
     
         4 . The method of  claim 1 , further comprising: 
 making fragments of said multiple copies of sense RNA.    
     
     
         5 . The method of  claim 4 , further comprising: 
 contacting said fragments of said multiple copies of sense RNA with a solid support comprising nucleic acid probes.    
     
     
         6 . The method of  claim 5 , further comprising: 
 detecting the presence or absence of hybridization of said fragments of said multiple copies of sense RNA to said nucleic acid probes on said solid support.    
     
     
         7 . The method of  claim 5 , wherein said solid support comprising nucleic acid probes is selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.  
     
     
         8 . The method of  claim 1 , further comprising: 
 creating an additional set of single-stranded DNA from said multiple copies of sense RNA;    synthesizing an additional set of double-stranded DNA from said additional set of single-stranded DNA; and    producing an additional set of multiple copies of sense RNA from said additional set of double-stranded DNA.    
     
     
         9 . The method of  claim 1 , wherein said fragments of a single-stranded DNA population are from about 30 nucleotides to about 3,000 nucleotides in length.  
     
     
         10 . The method of  claim 9 , wherein said fragments of a single-stranded DNA population are from about 30 nucleotides to about 750 nucleotides in length.  
     
     
         11 . The method of  claim 10 , wherein said fragments of a single-stranded DNA population are from about 30 nucleotides to about 150 nucleotides in length.  
     
     
         12 . The method of  claim 2 , wherein said RNA is isolated from an eukaryotic cell or tissue.  
     
     
         13 . The method of  claim 12 , wherein said eukaryotic cell or tissue is mammalian.  
     
     
         14 . The method of  claim 13 , wherein said mammalian cell or tissue is human.  
     
     
         15 . The method of  claim 2 , wherein said RNA is isolated from a source selected from the group consisting of dissected tissue, microdissected tissue, a tissue subregion, a tissue biopsy sample, a cell sorted population, a cell culture, and a single cell.  
     
     
         16 . The method of  claim 2 , wherein said RNA is isolated from a cell or tissue source selected from the group consisting of brain, liver, heart, kidney, lung, spleen, retina, bone, lymph node, endocrine gland, reproductive organ, blood, nerve, vascular tissue, and olfactory epithelium.  
     
     
         17 . The method of  claim 2 , wherein said RNA is isolated from a cell or tissue source selected from the group consisting of embryonic and tumorigenic.  
     
     
         18 . A proportionally amplified nucleic acid preparation comprising RNA obtained by the method of  claim 1 .  
     
     
         19 . A proportionally amplified nucleic acid preparation comprising RNA obtained by the method of  claim 8 .  
     
     
         20 . A gene expression monitoring system comprising a solid support, which comprises nucleic acid probes, and the proportionally amplified nucleic acid preparation of  claim 18 .  
     
     
         21 . A gene expression monitoring system comprising a solid support, which comprises nucleic acid probes, and the proportionally amplified nucleic acid preparation of  claim 19 .  
     
     
         22 . A nucleic acid detection system comprising the proportionally amplified nucleic acid preparation of  claim 18  immobilized to a solid support.  
     
     
         23 . A nucleic acid detection system comprising the proportionally amplified nucleic acid preparation of  claim 19  immobilized to a solid support.  
     
     
         24 . A method for the proportional amplification of nucleic acid, said method comprising: 
 creating fragments of a double-stranded DNA population; and    synthesizing multiple copies of said fragments of a double-stranded DNA population.    
     
     
         25 . The method of  claim 24 , wherein said double-stranded DNA population is produced from a nucleic acid population selected from the group consisting of one or more of the following: genomic DNA, cDNA, total RNA, poly(A) +  RNA, and oligonucleotides.  
     
     
         26 . The method of  claim 25 , wherein said poly(A) +  RNA is mRNA.  
     
     
         27 . The method of  claim 24 , further comprising: 
 labeling said multiple copies of said fragments of a double-stranded DNA population.    
     
     
         28 . The method of  claim 24 , further comprising: 
 contacting said multiple copies of said fragments of a double-stranded DNA population with a solid support comprising nucleic acid probes.    
     
     
         29 . The method of  claim 28 , further comprising: 
 detecting the presence or absence of hybridization of said multiple copies of said fragments of a double-stranded DNA population to said nucleic acid probes on said solid support.    
     
     
         30 . The method of  claim 28 , wherein said solid support comprising nucleic acid probes is selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.  
     
     
         31 . The method of  claim 24 , further comprising: 
 producing multiple copies of RNA from said multiple copies of said fragments of a double-stranded DNA population.    
     
     
         32 . The method of  claim 31 , further comprising: 
 contacting said multiple copies of RNA with a solid support comprising nucleic acid probes.    
     
     
         33 . The method of  claim 32 , further comprising: 
 detecting the presence or absence of hybridization of said multiple copies of RNA to said nucleic acid probes on said solid support.    
     
     
         34 . The method of  claim 32 , wherein said solid support comprising nucleic acid probes is selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.  
     
     
         35 . The method of  claim 24 , wherein said fragments of a double-stranded DNA population are from about 30 nucleotides to about 3,000 nucleotides in length.  
     
     
         36 . The method of  claim 35 , wherein said fragments of a double-stranded DNA population are from about 30 nucleotides to about 750 nucleotides in length.  
     
     
         37 . The method of  claim 36 , wherein said fragments of a double-stranded DNA population are from about 30 nucleotides to about 150 nucleotides in length.  
     
     
         38 . The method of  claim 25 , wherein said RNA is isolated from an eukaryotic cell or tissue.  
     
     
         39 . The method of  claim 38 , wherein said eukaryotic cell or tissue is mammalian.  
     
     
         40 . The method of  claim 39 , wherein said mammalian cell or tissue is human.  
     
     
         41 . The method of  claim 25 , wherein said RNA is isolated from a source selected from the group consisting of dissected tissue, microdissected tissue, a tissue subregion, a tissue biopsy sample, a cell sorted population, a cell culture, and a single cell.  
     
     
         42 . The method of  claim 25 , wherein said RNA is isolated from a cell or tissue source selected from the group consisting of brain, liver, heart, kidney, lung, spleen, retina, bone, lymph node, endocrine gland, reproductive organ, blood, nerve, vascular tissue, and olfactory epithelium.  
     
     
         43 . The method of  claim 25 , wherein said RNA is isolated from a cell or tissue source selected from the group consisting of embryonic and tumorigenic.  
     
     
         44 . A proportionally amplified nucleic acid preparation comprising DNA obtained by the method of  claim 24 .  
     
     
         45 . A proportionally amplified nucleic acid preparation comprising RNA obtained by the method of  claim 31 .  
     
     
         46 . A gene expression monitoring system comprising a solid support, which comprises nucleic acid probes, and the proportionally amplified nucleic acid preparation of  claim 44 .  
     
     
         47 . A gene expression monitoring system comprising a solid support, which comprises nucleic acid probes, and the proportionally amplified nucleic acid preparation of  claim 45 .  
     
     
         48 . A nucleic acid detection system comprising the proportionally amplified nucleic acid preparation of  claim 44  immobilized to a solid support.  
     
     
         49 . A nucleic acid detection system comprising the proportionally amplified nucleic acid preparation of  claim 45  immobilized to a solid support.  
     
     
         50 . A method for the proportional amplification of nucleic acid, said method comprising: 
 synthesizing multiple copies of a double-stranded DNA population; and    creating fragments of said multiple copies of a double-stranded DNA population.    
     
     
         51 . The method of  claim 50 , wherein said double-stranded DNA population is produced from a nucleic acid population selected from the group consisting of one or more of the following: genomic DNA, cDNA, total RNA, poly(A) +  RNA, and oligonucleotides.  
     
     
         52 . The method of  claim 51 , wherein said poly(A) +  RNA is mRNA.  
     
     
         53 . The method of  claim 50 , further comprising: 
 labeling said fragments of said multiple copies of a double-stranded DNA population.    
     
     
         54 . The method of  claim 50 , further comprising: 
 contacting said fragments of said multiple copies of a double-stranded DNA population with a solid support comprising nucleic acid probes.    
     
     
         55 . The method of  claim 54 , further comprising: 
 detecting the presence or absence of hybridization of said fragments of said multiple copies of a double-stranded DNA population to said nucleic acid probes on said solid support.    
     
     
         56 . The method of  claim 54 , wherein said solid support comprising nucleic acid probes is selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.  
     
     
         57 . The method of  claim 50 , further comprising: 
 producing multiple copies of RNA from said fragments of said multiple copies of a double-stranded DNA population.    
     
     
         58 . The method of  claim 51 , further comprising: 
 contacting said multiple copies of RNA with a solid support comprising nucleic acid probes.    
     
     
         59 . The method of  claim 58 , further comprising: 
 detecting the presence or absence of hybridization of said multiple copies of RNA to said nucleic acid probes on said solid support.    
     
     
         60 . The method of  claim 58 , wherein said solid support comprising nucleic acid probes is selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.  
     
     
         61 . The method of  claim 50 , wherein said fragments of said multiple copies of a double-stranded DNA population are from about 30 nucleotides to about 3,000 nucleotides in length.  
     
     
         62 . The method of  claim 61 , wherein said fragments of said multiple copies of a double-stranded DNA population are from about 30 nucleotides to about 750 nucleotides in length.  
     
     
         63 . The method of  claim 62 , wherein said fragments of said multiple copies of a double-stranded DNA population are from about 30 nucleotides to about 150 nucleotides in length.  
     
     
         64 . The method of  claim 51 , wherein said RNA is isolated from an eukaryotic cell or tissue.  
     
     
         65 . The method of  claim 64 , wherein said eukaryotic cell or tissue is mammalian.  
     
     
         66 . The method of  claim 65 , wherein said mammalian cell or tissue is human.  
     
     
         67 . The method of  claim 51 , wherein said RNA is isolated from a source selected from the group consisting of dissected tissue, microdissected tissue, a tissue subregion, a tissue biopsy sample, a cell sorted population, a cell culture, and a single cell.  
     
     
         68 . The method of  claim 51 , wherein said RNA is isolated from a cell or tissue source selected from the group consisting of brain, liver, heart, kidney, lung, spleen, retina, bone, lymph node, endocrine gland, reproductive organ, blood, nerve, vascular tissue, and olfactory epithelium.  
     
     
         69 . The method of  claim 51 , wherein said RNA is isolated from a cell or tissue source selected from the group consisting of embryonic and tumorigenic.  
     
     
         70 . A proportionally amplified nucleic acid preparation comprising DNA obtained by the method of  claim 50 .  
     
     
         71 . A proportionally amplified nucleic acid preparation comprising RNA obtained by the method of  claim 57 .  
     
     
         72 . A gene expression monitoring system comprising a solid support, which comprises nucleic acid probes, and the proportionally amplified nucleic acid preparation of  claim 70 .  
     
     
         73 . A gene expression monitoring system comprising a solid support, which comprises nucleic acid probes, and the proportionally amplified nucleic acid preparation of  claim 71 .  
     
     
         74 . A nucleic acid detection system comprising the proportionally amplified nucleic acid preparation of  claim 70  immobilized to a solid support.  
     
     
         75 . A nucleic acid detection system comprising the proportionally amplified nucleic acid preparation of  claim 71  immobilized to a solid support.