Method for generating transcriptionally active DNA fragments

Abstract

A method for producing transcriptionally active DNA molecules, comprising (PCR) amplification of said DNA molecule in the presence of a first DNA fragment (F1), second DNA fragment (F2), first primer (P1), a second primer (P2), a third primer (P3), and a fourth primer (P4) wherein: F1 comprises a promoter sequence; F2 comprises a terminator sequence; P1 is complementary to the 5′ end of F1; P2 is complementary to the 5′ end of F2; P3 comprises a first region complementary to the 3′ end of F1 and a second region complementary to the 5′ end of said DNA molecule; P4 comprises a first region complementary to the 3′ end of F2 and a second region complementary to the 3′ end of said DNA molecule.

What is claimed is:  
     
         1 . A method for amplifying a transcriptionally-active polynucleotide, comprising: 
 PCR-amplifying a first fragment of DNA with a first primer pair, wherein the first primer pair, upon such amplification, adds to first and second ends of the first fragment predetermined first and second regions of complementarity, to form a second DNA fragment having said first region of complementarity at a first end and a second region of complementarity at a second end of said second DNA fragment;    providing a promoter-containing sequence and a terminator-containing sequence, said promoter-containing sequence further including a region complementary to said first region of complementarity, and said terminator-containing sequence further including a region complementary to said second region of complementarity, wherein both said promoter-containing sequence and said terminator-containing sequence include an internal nucleotide capable of forming an A-T base pair immediately adjacent to said region of complementarity;    joining said promoter-containing sequence to said first end of said second DNA fragment and said terminator-containing sequence to said second end of said second DNA fragment to form said third DNA fragment; and    PCR-amplifying said third DNA fragment.    
     
     
         2 . The method of  claim 1 , wherein said joining comprises joining in the presence of polymerase said promoter-containing sequence to said first end of said second DNA fragment and said terminator-containing sequence to said second end of said second DNA fragment to form said third DNA fragment.  
     
     
         3 . The method of  claim 1 , wherein said promoter-containing sequence and said terminator-containing sequence further comprise a non-DNA, binding moiety capable of interacting with said second DNA fragment.  
     
     
         4 . The method of  claim 3 , wherein said non-DNA, binding moiety comprises a PNA molecule.