Process for preparation of full-length cDNA and anchor used for the same

Abstract

The present invention relates to a process for the preparation of full-length complementary DNA (cDNA). More particularly, the present invention is directed to a process for selective amplification of full-length cDNA, which comprises: i) a step for preparing a hybrid composed of a messenger RNA (mRNA) strand and a cDNA strand of which three (3) or four (4) deoxycitidinemono phosphate (dCMP) are combined at 3′ end, by treating mRNA with reverse transcriptase; separately from the above step, ii) a step for adenylating single strand anchor of which biotin or phosphate group is combined at 3′ end, and phosphate group is combined at 5′ end; iii) a step for ligating said adenylated single strand anchor to 3′ end of full-length cDNA strand of said cDNA/mRNA hybrid to select full-length cDNA/mRNA hybrid; and iv) a step for amplifying only the full-length cDNA/mRNA hybrid through polymerase chain reaction (PCR) which employs a primer of which base sequence is complementary to that of said anchor.

What is claimed is:  
     
         1 . A process for selective amplification of full-length cDNA, which comprises: 
 i) preparing a hybrid comprising an mRNA strand and a cDNA strand having three (3) or four (4) dCMPs at 3′ end by treating said mRNA with reverse transcriptase;    ii) adenylating a single strand anchor having a biotin or phosphate group at 3′ end and a phosphate group at 5′ end;    iii) ligating an adenylated single strand anchor obtained from step (ii) selectively to 3′ end of a full-length cDNA strand in a cDNA/mRNA hybrid obtained from step (i) to select a full-length cDNA/mRNA hybrid; and    iv) amplifying only said full-length cDNA strand through polymerase chain reaction by using a primer having a base sequence is complementary to that of said anchor.    
     
     
         2 . The process according to  claim 1 , wherein said reverse transcriptase is M-MLV.  
     
     
         3 . The process according to  claim 1 , wherein said RNA ligase is T4 RNA ligase.  
     
     
         4 . The process according to  claim 1 , which further comprises between step iii) and step iv): 
 a step for removing the residual single strand mRNA by using ribonuclease.    
     
     
         5 . A process for selective amplification of a part of cDNA or mRNA, which comprises: 
 i) preparing a hybrid comprising an mRNA strand and a cDNA strand having three (3) or four (4) dCMPs at 3′ end by treating said mRNA with reverse transcriptase;    ii) adenylating a single strand anchor having a biotin or phosphate group at 3′ end and a phosphate at 5′ end;    iii) ligating an adenylated single strand anchor obtained from step (ii) selectively to 3′ end of said cDNA strand of a cDNA/mRNA hybrid obtained from step (i) to select full-length cDNA/mRNA hybrid; and    iv) amplifying selectively a part of said full-length cDNA strand through polymerase chain reaction by using a gene-specific primer having a base sequence that is complementary to that of a target gene.    
     
     
         6 . A process for preparing a full-length cDNA, which comprises: 
 i) preparing a hybrid comprising an mRNA strand and a cDNA strand having three (3) or four (4) dCMPs at 3′ end by treating said mRNA with reverse transcriptase;    ii) adenylating a single strand anchor having a biotin or phosphate group at 3′ end and a phosphate group at 5′ end;    iii) ligating an adenylated single strand anchor obtained from step (ii) selectively to 3′ end of said cDNA strand of a cDNA/mRNA hybrid obtained from step (i) to select a full-length cDNA/mRNA hybrid;    iv) amplifying only said full-length cDNA strand through polymerase chain reaction by using a primer having a base sequence that is complementary to that of said anchor;    v) preparing a double strand cDNA that has specific cohesive ends, by cleaving a specific site of said anchor ligated to said full-length cDNA obtained from step (iv), using restriction enzyme;    vi) inserting said double strand cDNA containing a cohesive end into a vector using DNA ligase;    vii) transforming said vector containing said cDNA into a host cell;    viii) cloning said host cell in a large scale; and    ix) separating said full-length cDNA from said host cells, by cleaving said full-length cDNA from said vector with the restriction enzyme used in step v).    
     
     
         7 . An anchor that comprises one or more adenine group at its 5′ end.  
     
     
         8 . The anchor according to  claim 7 , wherein said anchor comprises a recognition site by a restriction enzyme selected from the group consisting of NotI, SmaI, XbaI and XhoI.  
     
     
         9 . The anchor according to  claim 7 , wherein said anchor comprises one or more regions having a base sequence that is complementary to that of a primer.  
     
     
         10 . The anchor according to  claim 7 , wherein said anchor is a single strand.  
     
     
         11 . The anchor according to  claim 8 , wherein said anchor is a single strand.  
     
     
         12 . The anchor according to  claim 9 , wherein said anchor is a single strand.  
     
     
         13 . The anchor according to  claim 8 , wherein said anchor comprises one or more regions having a base sequence that is complementary to that of a primer used for amplification of cDNA.  
     
     
         14 . A primer which specifically binds to an anchor comprising one or more thymine groups and three (3) to four (4) guanine groups at its 5′ end.  
     
     
         15 . The process according to  claim 5 , wherein said reverse transcriptase is M-MLV.  
     
     
         16 . The process according to  claim 5 , wherein said RNA ligate is T4 RNA ligase.  
     
     
         17 . The process according to  claim 5 , further comprising between step iii) and step iv): 
 a step for removing a residual single strand mRNA by using ribonuclease.    
     
     
         18 . The process according to  claim 17 , wherein said ribonuclease is ribonuclease A (RNase A).  
     
     
         19 . The process according to  claim 6 , wherein said reverse transcriptase is M-MLV.  
     
     
         20 . The process according to  claim 6 , wherein said RNA ligase is T4 RNA ligase.  
     
     
         21 . The process according to  claim 6 , further comprising a step for ligating an adenine group to said anchor.  
     
     
         22 . The process according to  claim 6 , further comprises between step iii) and step iv): 
 a step for removing a residual single strand mRNA using ribonuclease.    
     
     
         23 . The process according to  claim 22 , wherein said ribonuclease is RNase A.  
     
     
         24 . A process for obtaining a full-length cDNA, which comprises: 
 i) preparing a hybrid of an mRNA strand and a cDNA strand having three (3) or four (4) dCMPs at 3′ end by treating mRNA with reverse transcriptase;    ii) adenylating a single strand anchor having a biotin or phosphate group at 3′ end and a phosphate group at 5′ end; and    iii) ligating selectively said single strand anchor to 3′ end of said cDNA strand of a full-length cDNA/mRNA hybrid to select a full-length cDNA/mRNA hybrid.