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Detection of apolipoprotein B mRNA editing by peptide nucleic acid mediated PCR clamping
S Zhong1, N Y Nguyen, T L Eggerman
1Center for Biologics Evaluation and Research, Food and Drug Administration, NIH Building 29B, Room 2NN12, Bethesda, Maryland 20892, USA.
Abstract:
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.
Insights
A new PCR assay using PNA clamps can detect low levels of apolipoprotein B (apoB) mRNA editing. This method is more sensitive than current techniques for analyzing apoB-48 production.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Apolipoprotein B (apoB) mRNA editing results in a single base change, producing apoB-48.
- Current analysis of apoB mRNA editing uses RT-PCR primer extension, which is labor-intensive, radioactive, and lacks sensitivity for low editing levels.
Purpose of the Study:
- To develop a sensitive PCR-based assay for detecting the single base change in apoB mRNA editing.
- To improve the detection of low levels of apoB mRNA editing and apoB-48 production.
Main Methods:
- Development of a PCR-based assay utilizing Peptide Nucleic Acid (PNA) mediated PCR clamping.
- Combination of the PNA-mediated PCR clamping assay with the primer extension assay.
Main Results:
- The PNA-mediated PCR clamping assay can detect as low as 0.5% of the edited apoB mRNA form.
- Combining PNA clamping with primer extension resulted in an approximately 180-fold enrichment of the edited percentage.
Conclusions:
- PNA-mediated PCR clamping offers a highly sensitive method for detecting apoB mRNA editing.
- This novel assay improves upon existing methods for analyzing apoB-48 production, particularly at low editing levels.