Dual RT procedure for cDNA synthesis

Abstract

The present invention provides methods and compositions for the synthesis of long cDNA species. More particularly the present invention employs cycling between a low temperature and a high temperature reverse transcriptase activity to bypass the problem of secondary structures. Also described are methods of producing cDNA libraries and RT-PCR procedures.

What is claimed is:  
     
         1 . A method for the synthesis of cDNA comprising the steps of: 
 (a) providing a reaction mixture comprising a poly (A)+RNA, an oligonucleotide primer, dNTPs,    (b) incubating said reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating said reaction mixture at a normal temperature range to allow first strand synthesis;    (c) incubating said reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating said reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand;    (d) adding said first strand to a reaction mixture for the synthesis of a second strand complementary to said first strand wherein said second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of said second strand and incubating said reaction mixture under conditions to allow the formation of a double-stranded cDNA.    
     
     
         2 . The method of  claim 1 , wherein steps b and c are repeated.  
     
     
         3 . The method of  claim 2 , wherein said steps b and c are repeated once.  
     
     
         4 . The method of  claim 2 , wherein said steps b and c are repeated twice.  
     
     
         5 . The method of  claim 2 , wherein said steps b and c are repeated three times.  
     
     
         6 . The method of  claim 1 , wherein the reaction mixture of step (a) further comprises an RNase inhibitor.  
     
     
         7 . The method of  claim 1 , wherein the second strand synthesis reaction mixture of step (d) further comprises DEPC-treated H20.  
     
     
         8 . The method of  claim 1 , wherein the second strand synthesis reaction mixture of step (d) further comprises RNase H.  
     
     
         9 . The method of  claim 1 , further comprising the step of amplifying said double-stranded cDNA molecule of step (d).  
     
     
         10 . The method of  claim 9 , wherein the step of amplifying comprises PCR.  
     
     
         11 . The method of  claim 1 , wherein the temperature of step (b) is between about 37° C. and about 43° C.  
     
     
         12 . The method of  claim 1 , wherein the temperature of step (c) about 56° C. and about 95° C.  
     
     
         13 . The method of  claim 1 , wherein said processive reverse transcriptase is selected from the group consisting of Superscript™; AMV Reverse Transcriptase, M-MLV Reverse Transcriptase.  
     
     
         14 . The method of  claim 1 , wherein said thermostable reverse transcriptase is selected from the group consisting of Retrotherm™; Thermoscript™ and Tth reverse transcriptase.  
     
     
         15 . The method of  claim 1 , wherein said DNA polymerase is thermostable.  
     
     
         16 . The method of  claim 1 , wherein said DNA polymerase is selected from the group consisting of DNA Polymerase I, T4 DNA Polymerase, DNA Polymerase I Klenow fragment, PLATINUM taq™.  
     
     
         17 . The method of  claim 15 , wherein said thermostable DNA polymerase is selected from the group consisting of Tfl DNA Polymerase, Taq DNA Polymerase, Tli DNA Polymerase, Tth DNA Polymerase, Vent™, Deepvent™ and pfu.  
     
     
         18 . The method of  claim 1 , wherein said sample comprises between about 0.1 and picograms and 10 micrograms of polyA RNA.  
     
     
         19 . The method of  claim 1 , further comprising the step of adding linkers to said double stranded cDNA.  
     
     
         20 . The method of  claim 19 , wherein said linkers are added by blunt end ligation.  
     
     
         21 . The method of  claim 1 , wherein said reaction mixture comprises between 1 and 10 8  copies of said poly(A)+RNA.  
     
     
         22 . The method of  claim 1 , wherein said poly(A)+RNA is from a tumor.  
     
     
         23 . A method of increasing the length of cDNAs in a cDNA library comprising the steps of: 
 (a) providing a reaction mixture comprising a poly (A)+RNA, an oligonucleotide primer and dNTPs,    (b) incubating said reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating said reaction mixture at a normal temperature range to allow first strand synthesis;    (c) incubating said reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating said reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand;    (d) adding said first strand to a reaction mixture for the synthesis of a second strand complementary to said first strand wherein said second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of said second strand and incubating said reaction mixture under conditions to allow the formation of double-stranded cDNA; and    (e) amplifying said double-stranded cDNA of step (d)    wherein incubation at the temperatures in steps (c) inhibits the formation of secondary mRNA structures thereby resulting in cDNA species that are longer than in those produced in a normal temperature range.    
     
     
         24 . A method for the production of full length cDNAs comprising the steps of: 
 (a) providing a reaction mixture comprising a poly (A)+RNA, an oligonucleotide primer and dNTPs;    (b) incubating said reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating said reaction mixture at a normal temperature range to allow first strand synthesis;    (c) incubating said reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating said reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand;    (d) adding said first strand to a reaction mixture for the synthesis of a second strand complementary to said first strand wherein said second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of said second strand and incubating said reaction mixture under conditions to allow the formation of a double-stranded cDNA molecule, and    (e) amplifying said double-stranded cDNA molecule of step (d)    wherein the inhibition of secondary structure formation in step (b) allows the production of long cDNA moieties.    
     
     
         25 . The method of  claim 24 , wherein said cDNA moiety has a size of between about 0.5 kB and 20 kB.  
     
     
         26 . The method of  claim 24 , wherein said cDNA encodes a gene selected from the group consisting of XPC, CSA, XRCC3, XRCC2, XRCC9, ATM, ATR, RAD3, DNA-PK, ERCC1, XPA, XPB, XPC, XPD, XPF, XPG, CSB and HHR23B  
     
     
         27 . The method of  claim 24 , wherein said cDNA encodes a gene related to colorectal carcinoma.  
     
     
         28 . The method of  claim 27 , wherein said colorectal carcinoma is hereditary colorectal carcinoma.  
     
     
         29 . The method of  claim 27 , wherein said colorectal carcinoma is sporadic colorectal carcinoma.  
     
     
         30 . The method of  claim 28 , wherein said gene is selected from the group consisting of hMSH2, hMLH1, hPMS1, hPMS2 and GTBP.  
     
     
         31 . The method of  claim 29 , wherein said gene is selected from the group consisting of transforming growth factor b type II receptor, insulin-like growth factor II receptor, BAX and μ2-microglobulin.  
     
     
         32 . A method for synthesizing long cDNA moieties comprising the steps of 
 (a) providing a reaction mixture comprising a poly (A)+RNA, an oligonucleotide primer and dNTPs,    (b) incubating said reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating said reaction mixture at a normal temperature range to allow first strand synthesis;    (c) incubating said reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating said reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand;    (d) adding said first strand to a reaction mixture for the synthesis of a second strand complementary to said first strand wherein said second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of said second strand and incubating said reaction mixture under conditions to allow the formation of a double-stranded cDNA, and    e) amplifying said double-stranded cDNA molecule of step (d)    wherein the inhibition of secondary structure formation in step (b) allows the production of cDNA moieties that are longer than those obtained when such secondary structure formation is not inhibited.    
     
     
         33 . A method for producing a library of cDNA species from a tumor comprising the steps of: 
 (a) providing a reaction mixture comprising a poly (A)+RNA extracted from said tumor, an oligonucleotide primer and dNTPs;    (b) incubating said reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating said reaction mixture at a normal temperature range to allow first strand synthesis;    (c) incubating said reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating said reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand;    (d) adding said first strand to a reaction mixture for the synthesis of a second strand complementary to said first strand wherein said second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of said second strand and incubating said reaction mixture under conditions to allow the formation of a double-stranded cDNA, and    (e) amplifying said double-stranded cDNA molecule of step (d)    (f) inserting said cDNA into an appropriate vector.    
     
     
         34 . The method of  claim 33 , wherein said tumor is a colorectal tumor.