Detection of apolipoprotein B mRNA editing by peptide nucleic acid mediated PCR clamping

Apolipoprotein B (apoB) mRNA editing leads to a single base change in its m RNA and the production of apoB-48. Currently, the degree of apoB mRNA editi ng is analyzed by the RT-PCR primer extension method. While this method is quantitative, it is labor intensive, utilizes radioactivity for labeling an d may not be sensitive enough to discriminate between low levels of editing and inherent assay background levels. Peptide nucleic acid (PNA) oligonucl etides have been used in single point mutation detection through PCR. clamp ing. In the present work, we developed a PCR based assay which can detect t he single base change responsible for the apoB-48 production. We found that as low as 0.5% of the edited form can be clearly detected by PNA mediated PCR clamping. When combined with the primer extension assay, an approximate ly 180-fold enrichment of the edited percentage is observed, reflecting sel ected PCR amplification of templates containing the edited base. (C) 1999 A cademic Press.