Lab Practical Notes on p53 Mutation Detection

Learning Outcomes

• Understand principles & practice of gel electrophoresis and protein separation.

• Gain knowledge on Western blotting procedure.

• Use PCR to amplify human genomic DNA.

• Identify Restriction Fragment Length Polymorphism (RFLP) in samples.

• Recognize differences in fragment sizes.

• Exploit known mutations for diagnostic assay design.

• Learn basic molecular biology techniques.

Background on p53 Mutations

• p53 (TP53) is a tumor suppressor gene, frequently mutated in cancers.

• Acts as a "guardian of the genome", preventing cell division when DNA is damaged.

• Known for functions like cell cycle arrest, DNA repair, and induction of apoptosis in response to stress.

• Over 30,500 mutations recorded in UMD TP53 mutation database.

• c.742C>T (p.R248W) is a common mutation leading to complete loss of function and can be detected diagnostically via restriction enzyme analysis.

Techniques Used

Gel Electrophoresis & Western Blotting

• Day 1: Gel Electrophoresis

  • Load and separate protein samples, perform staining, and transfer proteins to a membrane.

• Day 2: Western Blotting

  • Apply secondary antibodies and develop signal for detection.

Protocols and Safety Guidelines

• Wear gloves and handle chemicals with care.

• Be familiar with reagents (e.g., loading buffer, antibodies, gel solutions) and equipment (e.g., gel tanks, transfer apparatus).

Protocol Steps for SDS-PAGE Gel Electrophoresis

1. Prepare and load samples into the gel.

2. Run gel at 150V for 50 minutes.

3. Perform Turbo Blot Transfer to transfer proteins onto PVDF membrane.

4. Stain and visualize using Coomassie blue.

Analysis Considerations

• Evaluate protein expression changes between samples using size comparison and overall profile similarity.

• Contemplate the impact of mutations on molecular weight and protein functionality.

• Answer critical questions about molecular weight determination and setup order for electroelution in Western blotting.