cDNA libraries and methods for their production

Abstract

The present invention provides new methods of synthesizing cDNAs, methods of verifying full-length cDNAs, methods of producing cDNA libraries enriched for full-length inserts, and the like.

What is claimed is:  
     
         1 . A method for producing a full-length cDNA, comprising the steps of: 
 contacting a ribonucleic acid to an agent that inhibits formation of secondary structure;    contacting the ribonucleic acid molecule with a primer and a polymerase to form a first-strand synthesis mixture, wherein said polymerase comprises 3′-5′ exonuclease activity;    exposing the first-strand synthesis mixture to conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a hybrid molecule comprising said ribonucleic acid molecule hybridized to a first deoxyribonucleic acid molecule comprising the first primer is produced; and    converting said hybrid molecule to a double-stranded deoxyribonucleic acid molecule.    
     
     
         2 . The method of  claim 1 , wherein the agent is present in the transcription mixture and is selected from the group consisting of dimethyl sulfoxide (DMSO) at a concentration of about 5-20%, glycerol at a concentration of about 5-40%, formamide at a concentration of about 5-10%, and betaine at a concentration of about 0.5-2.2 M.  
     
     
         3 . The method of  claim 1 , wherein the reverse transcriptase is selected from the group consisting of: Pfu DNA polymerase, Tma DNA polymerase, Tne polymerase, and Tli DNA polymerase.  
     
     
         4 . A method of isolating a full-length cDNA, said method comprising: 
 contacting a ribonucleic acid molecule with a first primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a hybrid molecule comprising said ribonucleic acid molecule hybridized to a first deoxyribonucleic acid molecule comprising the first primer is produced;    contacting the hybrid molecule with a detectably labeled oligo-dV primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, wherein said primer results in additional production of hybrid sequences provided first hybrid molecule does not extend to the 5′ end of the ribonucleic acid;    isolating a hybrid molecule that does not contain the detectable label; and    converting said unlabelled hybrid molecule to a double-stranded deoxyribonucleic acid molecule.    
     
     
         5 . The method of  claim 4 , wherein said single-stranded ribonucleic acid molecule is an mRNA.  
     
     
         6 . The method of  claim 4 , wherein the first primer is an oligo-dT primer.  
     
     
         7 . The method of  claim 4 , wherein the first primer is a random primer.  
     
     
         8 . The method of  claim 4 , wherein the oligo-dV primer is labeled with biotin.  
     
     
         9 . The method of  claim 4 , wherein the oligo-dV primer is radioactively labeled.  
     
     
         10 . The method of  claim 4 , wherein the oligo-dV primer is comprised of 4-12 nucleotides.  
     
     
         11 . The method of  claim 4 , wherein said method further comprises introducing said double-stranded cDNA molecule into a vector.  
     
     
         12 . A method of identifying a full-length first strand cDNA, the method comprising the steps of: 
 contacting a ribonucleic acid molecule hybridized to a deoxyribonucleic acid molecule with a detectably labeled oligo-dV primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, wherein said primer results in additional production of hybrid sequences provided the deoxyribonucleic acid does not extend to the 5′ end of the ribonucleic acid; and    detecting label in the cDNA;    wherein label in the cDNA is indicative of a non-full-length cDNA.    
     
     
         13 . The method of  claim 12 , further comprising: 
 contacting a ribonucleic acid molecule with a first primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur to produce a hybrid molecule comprising said ribonucleic acid molecule hybridized to a deoxyribonucleic acid molecule.    
     
     
         14 . A method for producing a cDNA library from a sample of mRNA molecules, said method comprising: 
 contacting said mRNA molecules with a first primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a population of hybrid molecules comprising said mRNA molecules hybridized to a population of cDNA molecules comprising said first primer is produced;    isolating the population of hybrid molecules;    contacting said isolated hybrid molecules with a detectably labeled oligo-dV primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, wherein said primer results in additional production of hybrid sequences provided the first hybrid molecule does not extend to the 5′ end of the ribonucleic acid;    converting said population of hybrid molecules to a double-stranded deoxyribonucleic acid molecule;    separating said population of double-stranded cDNA molecules into labeled double-stranded cDNA molecules and unlabelled double-stranded cDNA molecules; and    introducing each unlabelled double-stranded cDNA molecules into a vector to produce a library enriched in full-length cDNAs.    
     
     
         15 . The method of  claim 14 , wherein the first primer is oligo-dT primer.  
     
     
         16 . The method of  claim 14 , wherein the first primer is a random primer.  
     
     
         17 . The method of  claim 14 , wherein said method further comprises introducing the population of double-stranded cDNA molecules into vectors.  
     
     
         18 . The method of  claim 14 , wherein said method further comprises selectively cloning only the 5′ end of a full-length cDNA to produce a cDNA library comprising the 5′ ends of expressed genes.  
     
     
         19 . A method for producing a cDNA library from a sample of mRNA molecules, said method comprising: 
 contacting said mRNA molecules with an agent that inhibits formation of secondary structure;    contacting said mRNA molecules with a primer and a polymerase to form a first-strand synthesis mixture, wherein said polymerase comprises 3′-5′ exonuclease activity;    exposing the first-strand synthesis mixture to conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur whereby a population of hybrid molecules comprising said mRNA molecules hybridized to a population of cDNA molecules comprising said first primer is produced;    isolating the population of hybrid molecules;    contacting said isolated hybrid molecules with a detectably labeled oligo-dV primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, wherein said primer results in additional production of hybrid sequences provided the first hybrid molecule does not extend to the 5′ end of the ribonucleic acid;    converting said population of hybrid molecules to a double-stranded deoxyribonucleic acid molecule;    separating said population of double-stranded cDNA molecules into labeled double-stranded cDNA molecules and unlabelled double-stranded cDNA molecules; and    introducing each unlabelled double-stranded cDNA molecules into a vector to produce a library enriched in full-length cDNAs.