Lab 5: Genetic Disease and Molecular Diagnosis

Burkitt Lymphoma

• A fast-growing cancer and a type of non-Hodgkin lymphoma (NHL)
• Characterized by many genetic changes that cause cells to act differently than normal cells
• Three types:
  • Endemic Burkitt lymphoma: most often develops in children living in Africa
  • Sporadic Burkitt lymphoma: affects children worldwide and is very rare in adults
  • Immunodeficiency-related Burkitt lymphoma: develops in people with immune system problems

Diagnosis of Burkitt Lymphoma

• Biopsy: examining a sample of tissue under a microscope to confirm or rule out Burkitt lymphoma
• Molecular diagnosis:
  • Cytogenetic findings: assessing gross genomic changes of lymphoma cells, including ploidy, deletions, translocations, and marker chromosomes
  • Karyotyping: identifying the partner chromosome of the c-MYC translocation
  • Gene expression: analyzing molecular alterations leading to c-MYC dysregulation and its effect on prognosis and diagnosis

Molecular Diagnosis of c-MYC Gene Translocation

• RNA isolation: using TRIzol™ reagent to isolate total RNA from patients and healthy controls
• RNA quantification: using nanodrop A260/280 readings to ensure purity
• Reverse Transcription-Quantitative PCR (RT-qPCR): using the GoTaq® 1-Step RT-qPCR System kit to analyze RNA
• Synthesis of complementary DNA (cDNA) from mRNA: using GoScript™ RT Mix for 1-Step RT-qPCR
• Primer sequences:
  • c-myc forward primer: 5′-TAC CCT CTC AAC GAC AGC AG-3′
  • c-myc reverse primer: 5′-TCT TGA CAT TCT CCT CGG TG-3′
  • β-actin forward primer: 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′
  • β-actin reverse primer: 5′CAG CGG AAC CGC TCA TTG CCA ATG G-3′ (control gene)

Real-Time PCR Reaction

• Steps:
  • cDNA Synthesis: 37°C for 15 minutes
  • Initial Denaturation: 95°C for 5 minutes
  • Denaturation: 95°C for 30 seconds
  • Annealing: 60°C for 30 seconds
  • Extension: 72°C for 30 seconds
  • Melt curve: 65-90°C
  • Holding: 4°C