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 mRNA and the production of apoB-48. Currently, the degree of apoB mRNA editing is analyzed by the RT-PCR primer extension method. While this method is quantitative, it is labor intensive, utilizes radioactivity for labeling and may not be sensitive enough to discriminate between low levels of editing and inherent assay background levels. Peptide nucleic acid (PNA) oligonucletides have been used in single point mutation detection through PCR clamping. In the present work, we developed a PCR based assay which can detect the 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 approximately 180-fold enrichment of the edited percentage is observed, reflecting selected PCR amplification of templates containing the edited base.
Zhong
S
S
Center for Biologics Evaluation and Research, Food and Drug Administration, NIH Building 29B, Room 2NN12, Bethesda, Maryland 20892, USA.
Nguyen
N Y
NY
Eggerman
T L
TL
D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020135:Peptide Nucleic Acids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D017393:RNA Editing / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D017931:DNA Primers; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger