Method for the 5'-cap-dependent amplification of cDNAs

Abstract

The invention concerns a method for the modification, cloning and amplification of cDNAs which are complete at their 5′ end which is essentially characterized in that the first strand cDNA synthesis is carried out in the presence of manganese 2+ ions or manganese 2+ is added as an additive at a later time. The CAP structure at the 5′ end of the reversely transcribed mRNA triggers the attachment of deoxy-cytosines to the 3′ end of the cDNA with high efficiency. In a preferred embodiment a controlled ribonucleotide tailing is carried out with the aid of terminal transferase following the first strand cDNA synthesis.

What is claimed is:  
     
         1 . A method for the modification, cloning or amplification of a cDNA, wherein the first strand cDNA synthesis is carried out in the presence of magnesium 2+  ions and manganese 2+  ions.  
     
     
         2 . A method for the modification, cloning or amplification of a cDNA, wherein the cDNA is incubated in the presence of manganese 2+  ions directly after the first strand cDNA synthesis.  
     
     
         3 . The method of  claim 1 , wherein the concentration of manganese 2+  ions is at least 1 mM but at most 20 mM during the cDNA synthesis.  
     
     
         4 . The method of  claim 1 , wherein after the cDNA synthesis, the cDNA is reacted with a ribonucleotide triphosphate or a modified nucleotide triphosphate in the absence of terminal transferase.  
     
     
         5 . The method of  claim 4 , wherein the product of the reaction is linked to a further double-stranded nucleic acid molecule which has a 3′ over-hanging end and is complementary to the 3′ end of the product of the reaction.  
     
     
         6 . The method of  claim 5 , wherein the further nucleic acid molecule is a DNA vector or an adaptor molecule.  
     
     
         7 . The method of  claim 5 , wherein the further nucleic acid molecule is ligated to the 3′ end of the product of the reaction.  
     
     
         8 . The method of  claim 7 , wherein the ligation is carried out in the presence of 5 to 10 vol % DMSO.  
     
     
         9 . The method of  claim 8 , wherein the specific ligation is carried out with an efficiency of more than 90%.  
     
     
         10 . The method of  claim 7 , wherein the amplification is carried out using a primer provided with an immobilizable group.  
     
     
         11 . The method of  claim 7 , wherein the double-stranded DNA adaptor molecule is provided with an immobilizable group.  
     
     
         12 . The method of  claim 10 , wherein the amplification reaction is carried out several times.  
     
     
         13 . The method of  claim 7 , wherein a nested PCR is carried out.  
     
     
         14 . The method of  claim 10 , wherein the amplification product is directly sequenced.  
     
     
         15 . A mixture of double-stranded DNA adaptors containing 3′ overhangs composed of 5′-dT 3-4 dG 3 -3′.  
     
     
         16 . First strand cDNA characterized by a non mRNA template-coded 3′ end composed of 5′-dC 3-4 rA 3-4 -3′.