## Investigation of Choice for CFTR Mutation Detection ### Why PCR + Sequencing is Gold Standard **Key Point:** PCR followed by direct DNA sequencing is the gold standard for detecting point mutations and small deletions like ΔF508 in the CFTR gene because it: - Amplifies the target region exponentially (2^n cycles) - Provides high sensitivity and specificity - Allows direct visualization of the mutation at the nucleotide level - Is rapid, cost-effective, and widely available ### Comparison of Blotting and PCR Techniques | Technique | Target | Sensitivity | Best For | Limitations | |-----------|--------|-------------|----------|-------------| | **PCR + Sequencing** | DNA | Very high | Point mutations, small indels, CFTR screening | Requires optimization | | **Southern Blot** | DNA | Moderate | Large deletions, rearrangements | Slow, requires radioactivity, less sensitive | | **Northern Blot** | mRNA | Moderate | Gene expression analysis | Does not detect DNA mutations directly | | **Western Blot** | Protein | Low for mutation detection | Protein abnormalities | Indirect, protein-level only | ### Clinical Pearl **High-Yield:** The ΔF508 deletion accounts for ~70% of CF mutations worldwide. PCR can be designed with allele-specific primers to detect this common mutation rapidly, or multiplex PCR panels can screen for the most common CFTR mutations in a single reaction. ### Mechanism of PCR Advantage 1. **Amplification:** PCR generates millions of copies of the target CFTR exon 10 region 2. **Direct sequencing:** Sanger or next-generation sequencing reads the amplified product 3. **Mutation visualization:** The ΔF508 deletion (3 bp loss) is immediately apparent as a shorter fragment or frameshift in the sequence **Mnemonic:** **PCR-SEQ** = **P**recision **C**FTR **R**esolution via **S**equencing for **E**xact **Q**uality [cite:Robbins 10e Ch 5]
Sign up free to access AI-powered MCQ practice with detailed explanations and adaptive learning.