MOLECULAR-CLONING AND SEQUENCING OF AN INTRON OF HER-2 NEU (ERBB2) GENE

This report documents the cloning and sequencing of a previously unkno wn intron of the Her-2/neu gene and compares three different cloning s trategies for efficient PCR cloning. Although successful results have previously been documented using blunt-end ligation strategies, the TA cloning system (Invitrogen, CA) offered the best result in our hands using a PCR-amplified DNA fragment of Her-2/neu (ERBB2) gene containin g an unreported intron. The TA cloning system is easy to manipulate an d the cloned DNA can be easily sequenced without further subcloning in to the M13 vector. Additionally, this cloning strategy does not requir e (i) any prior selection of restriction sites during primer design, ( ii) post PCR restriction digestion, and/or (iii) gel purification of P CR-amplified DNA. The newly identified and sequenced DNA of the Her-2/ neu intron (GenBank Accession No. M95667) may help in designing primer s for the analysis of the Her-2/neu gene in biological specimens. Ther efore, we recommend the TA cloning system as the preferred choice for cloning any DNA fragments generated by PCR.