Methods of small sample amplification

Abstract

The present invention relates to the amplification of nucleic acids, preferably from mRNA. A primer and promoter are added to a target sequence to be amplified and then the target is amplified in an in vitro transcription reaction and the product of this reaction is used as template for subsequent rounds of amplification.

1 . A method for the amplification of nucleic acid, said method comprising: 
 (a) contacting mRNA having a poly d(A) tail with a primer comprising poly d(T) and a second sequence; and,    generating a first cDNA strand from the mRNA strand by extending the primer by reverse transcriptase and the appropriate nucleotides under the appropriate conditions, which creates a RNA:DNA duplex;    (b) denaturing the RNA:DNA duplex;    (c) forming a double stranded DNA;    (d) adding a promoter to the DNA by hybridizing a promoter to the single stranded DNA;    (e) forming a double stranded DNA promoter region by adding the appropriate reagents;    (f) producing multiple copies of RNA comprising at least part of said second sequence from the DNA strand comprising the promoter;    (g) contacting said multiple copies of RNA with random primers; and,    generating a first DNA strand from the RNA by extending the primer by reverse transciptase, which creates a RNA:DNA duplex;    (h) denaturing the RNA:DNA duplex; and,    adding a promoter to the DNA by hybridizing a promoter to the single stranded DNA;    (i) forming a double stranded DNA promoter region by adding the appropriate reagents; and,    (j) producing multiple copies of RNA from the DNA strand comprising the promoter    
     
     
         2 . The method of  claim 1  wherein steps (g-j) are repeated at least once.  
     
     
         3 . The method of  claim 1  wherein said amplification is proportional.  
     
     
         4 . The method of  claim 1 , wherein at least one of the enzymes used may be thermostable.  
     
     
         5 . The method of  claim 1  wherein said nucleic acid is selected from the group consisting of genomic DNA, cDNA, total RNA, poly(A) +  RNA, and oligonucleotides.  
     
     
         6 . The method of  claim 1 , wherein amplification is accomplished using a promoter that is blocked from extending in a 3′ direction.  
     
     
         7 . The method of  claim 1  wherein said poly(A) +  RNA is mRNA.  
     
     
         8 . The method of  claim 1  further comprising: 
 contacting said multiple copies of RNA produced in step (j) with a solid support comprising nucleic acid probes.    
     
     
         9 . The method of  claim 8  further comprising: 
 detecting the presence or absence of hybridization of said multiple copies of RNA produced in step (j) to said nucleic acid probes on said solid support.    
     
     
         10 . The method of  claim 8  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.  
     
     
         11 . The method of  claim 1  wherein said nucleic acid is isolated from an eukaryotic cell or tissue.  
     
     
         12 . The method of  claim 11 , wherein said eukaryotic cell or tissue is mammalian.  
     
     
         13 . The method of  claim 12  wherein said mammalian cell or tissue is human.  
     
     
         14 . The method of  claim 1  wherein said nucleic acid 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.  
     
     
         15 . The method of  claim 1  wherein said nucleic acid 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.  
     
     
         16 . The method of  claim 1  wherein said nucleic acid is isolated from a cell or tissue source selected from the group consisting of embryonic and tumorigenic.  
     
     
         17 . An amplified nucleic acid preparation comprising RNA obtained by the method of  claim 1 .  
     
     
         18 . An amplified nucleic acid preparation comprising RNA obtained by the method of  claim 6 .  
     
     
         19 . A gene expression monitoring system comprising a solid support, which comprises nucleic acid probes and the amplified nucleic acid preparation of  claim 1 .  
     
     
         20 . A gene expression monitoring system comprising a solid support, which comprises nucleic acid probes and the amplified nucleic acid preparation of  claim 6 .  
     
     
         21 . A nucleic acid detection system comprising the amplified nucleic acid preparation of  claim 1  immobilized to a solid support.  
     
     
         22 . A nucleic acid detection system comprising the amplified nucleic acid preparation of  claim 6  immobilized to a solid support.  
     
     
         23 . The method of  claim 1  wherein the method involves the use of an automated machine.  
     
     
         24 . The method of  claim 23  wherein said automated machine is selected from the group consisting of a thermocycler, an integrated reaction device, and a robotic delivery system.  
     
     
         25 . A kit for the amplification of nucleic acids, wherein said kit comprises a container, instructions for use, a promoter which comprises a poly d(T) sequence operably linked to a second sequence, a promoter comprising the second sequence or its equivalent and a sequence that will complete a promoter when annealed to a target and formed into a double stranded DNA.  
     
     
         26 . The kit of  claim 25  wherein the second sequence is a unique nucleic acid sequence which does not have a promoter function.  
     
     
         27 . The kit of  claim 26  wherein the unique sequence is blocked from extension in the 3′ direction.  
     
     
         28 . The kit of  claim 26  wherein the second sequence comprises a portion of a promoter.  
     
     
         29 . The kit of  claim 25  wherein the promoter is blocked from extension in the 3′ direction.  
     
     
         30 . A method for the amplification of nucleic acid, said method comprising: 
 (a) contacting mRNA having a poly d(A) tail with a primer comprising poly d(T) and a second sequence comprising a functional promoter sequence; and,    generating a first cDNA strand from the mRNA strand by extending the first primer by reverse transcriptase and the appropriate nucleotides under the appropriate conditions, which creates a RNA:DNA duplex;    (b) denaturing the RNA:DNA duplex;    (c) contacting the DNA with random primers and forming a double stranded DNA with a functional promoter region;    (d) producing multiple copies of RNA from the DNA strand comprising the promoter;    (e) contacting said multiple copies of RNA with random primers; and,    generating a first DNA strand from the RNA strand by extening the primer by reverse transcriptase, which creates a RNA:DNA duplex;    (f) denaturing the RNA:DNA duplex; and, adding a primer comprising poly d(T) and a promoter sequence;    (g) forming a double stranded DNA;    (h) forming a double stranded DNA promoter region by adding the appropriate reagents; and,    (i) producing multiple copies of RNA from the DNA strand comprising the promoter    
     
     
         31 . The method of  claim 30 , wherein steps (e-i) are repeated at least once.  
     
     
         32 . The method of  claim 30 , wherein said amplification is proportional.  
     
     
         33 . The method of  claim 30 , wherein said nucleic acid is selected from the group consisting of genomic DNA, cDNA, total RNA, poly(A) +  RNA, and oligonucleotides.  
     
     
         34 . The method of  claim 30 , wherein the second sequence comprises a partial sequence for the RNA promoter.  
     
     
         35 . The method of  claim 30 , wherein said poly(A) +  RNA is mRNA.  
     
     
         36 . The method of  claim 30 , further comprising: 
 contacting said multiple copies of RNA produced in step (i) with a solid support comprising nucleic acid probes.    
     
     
         37 . The method of  claim 36  further comprising: 
 detecting the presence or absence of hybridization of said multiple copies of RNA produced in step (i) to said nucleic acid probes on said solid support.    
     
     
         38 . The method of  claim 36  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.  
     
     
         39 . The method of  claim 30  wherein said nucleic acid is isolated from an eukaryotic cell or tissue.  
     
     
         40 . The method of  claim 39 , wherein said eukaryotic cell or tissue is mammalian.  
     
     
         41 . The method of  claim 40  wherein said mammalian cell or tissue is human.  
     
     
         42 . The method of  claim 30  wherein said nucleic acid 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.  
     
     
         43 . The method of  claim 30  wherein said nucleic acid 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.  
     
     
         44 . The method of  claim 30  wherein said nucleic acid is isolated from a cell or tissue source selected from the group consisting of embryonic and tumorigenic.  
     
     
         45 . An amplified nucleic acid preparation comprising RNA obtained by the method of  claim 30 .  
     
     
         46 . A gene expression monitoring system comprising a solid support, which comprises nucleic acid probes and the amplified nucleic acid preparation of  claim 30 .  
     
     
         47 . A nucleic acid detection system comprising the amplified nucleic acid preparation of  claim 30  immobilized to a solid support.  
     
     
         48 . The method of  claim 30  wherein the method involves the use of an automated machine.  
     
     
         49 . The method of  claim 48  wherein said automated machine is selected from the group consisting of a thermocycler, an integrated reaction device, and a robotic delivery system.  
     
     
         50 . A kit for the amplification of nucleic acids, wherein said kit comprises a container, instructions for use, a primer which comprises a poly d(T) sequence operably linked to a promoter sequence.  
     
     
         51 . The kit of  claim 50  further comprising a second primer comprising a poly d(T) sequence operably linked to a promoter sequence, wherein said second primer is blocked from extension in the 3′ direction.  
     
     
         52 . A method for the amplification of nucleic acid, said method comprising: 
 (a) contacting mRNA having a poly d(A) tail with a primer comprising poly d(T) operably linked to a functional promoter sequence; and,    generating a first cDNA strand from the mRNA strand by extending the first primer by reverse transcriptase and the appropriate nucleotides under the appropriate conditions, which creates a RNA:DNA duplex;    (b) denaturing the RNA:DNA duplex;    (c) contacting the DNA with random primers and forming a double stranded DNA with a functional promoter region;    (d) producing multiple copies of RNA from the DNA strand comprising the promoter;    (e) contacting said multiple copies of RNA with random primers; and, generating a first DNA strand from the RNA strand by extening the primer by reverse transcriptase, which creates a RNA:DNA duplex;    (f) denaturing the RNA:DNA duplex; and, adding a primer comprising poly d(T) and a promoter sequence wherein the primer is blocked from extending in a 3′ direction;    (g) forming a double stranded DNA promoter region by adding the appropriate reagents; and,    (h) producing multiple copies of RNA from the DNA strand comprising the promoter.    
     
     
         53 . An amplified nucleic acid preparation comprising RNA obtained by the method of  claim 52 .  
     
     
         54 . A gene expression monitoring system comprising a solid support, which comprises nucleic acid probes and the amplified nucleic acid preparation of  claim 52 .  
     
     
         55 . A nucleic acid detection system comprising the amplified nucleic acid preparation of  claim 52  immobilized to a solid support.