Use of T7 gene 6 exonuclease and phosphorothioated primers for the manipulation of HIV-1 infectious clones

A method is described for the efficient substitution, deletion or insertion of any desired DNA sequence into any viral infectious clones without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction combined with the resistance of 2'-deoxynucleotides 5'-O-(1-thiotriphosphate) dNTPs [S] bonds (phosphorothiate bonds) to the 5'-3' double strand specific T7 gene 6 exonuclease (T7 Exo) digestion. Primers used to amplify the DNA target regions being manipulated present three phosphorothioate bonds from the fifteenth base at the 5' end. The enzyme activity was shown to be completely inhibited by the presence of more than one phosphorothioate residue at the 5' end of the DNA molecules. When the amplification products are submitted to the exonuclease digestion the hydrolytic T7 fro activity generates a short single strand DNA tail which contains the nucleotide integrity of the 3' strand. Since the ends of two independently amplified products overlap they can regenerate a stable recombinant structure when further combined in the same reaction tube in the presence of T4 DNA ligase. This new method can be used for manipulating an HIV-1 full-length clone belonging to subtype D replacing the env (gp120) gene for an F subtype sequence. (C) 1998 Elsevier Science B.V. All rights reserved.