Molecular cloning and sequencing of an intron of Her-2/neu (ERBB2) gene.
This report documents the cloning and sequencing of a previously unknown intron of the Her-2/neu gene and compares three different cloning strategies 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 containing an unreported intron. The TA cloning system is easy to manipulate and the cloned DNA can be easily sequenced without further subcloning into the M13 vector. Additionally, this cloning strategy does not require (i) any prior selection of restriction sites during primer design, (ii) post PCR restriction digestion, and/or (iii) gel purification of PCR-amplified DNA. The newly identified and sequenced DNA of the Her-2/neu neu intron (GenBank Accession No. M95667) may help in designing primers for the analysis of the Her-2/neu gene in biological specimens. Therefore, we recommend the TA cloning system as the preferred choice for cloning any DNA fragments generated by PCR.
Sarkar
F H
FH
Department of Pathology, Wayne State University School of Medicine, Detroit, MI.
Ball
D E
DE
Li
Y W
YW
Crissman
J D
JD
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D007438:Introns; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases
D009856:Oncogene Proteins, Viral; D018719:Erb-b2 Receptor Tyrosine Kinases