Lab 5: Genetic Disease and Molecular Diagnosis

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Questions and Answers

What is the type of cancer that Burkitt lymphoma is a part of?

Non-Hodgkin lymphoma (correct)

Melanoma

Leukemia

Hodgkin lymphoma

In which region of the world does endemic Burkitt lymphoma most often develop in children?

South America

Africa (correct)

Asia

Europe

What is the purpose of a biopsy in diagnosis of Burkitt lymphoma?

To determine the severity of the disease

To identify the genetic changes in the cells

To rule out Burkitt lymphoma

To confirm the presence of Burkitt lymphoma (correct)

What is the name of the gene translocation that is assessed in molecular diagnosis of Burkitt lymphoma?

c-MYC translocation Signup and view all the answers

What is the term for the study of the gross genomic changes of lymphoma cells?

Karyotyping Signup and view all the answers

Which of the following groups of people are at risk of developing immunodeficiency-related Burkitt lymphoma?

People who have had an organ transplant and need to take immunosuppressive drugs Signup and view all the answers

What is the advantage of using karyotyping in genetic diagnosis?

Identifying the partner chromosome of the c-MYC translocation Signup and view all the answers

What is the purpose of using TRIzol™ reagent in RNA isolation?

To isolate total RNA from patients and healthy controls Signup and view all the answers

What is the function of SYBR®Green I dye in the GoTaq®1-Step RT-qPCR System kit?

To bind to double-stranded DNA Signup and view all the answers

What is the purpose of the GoTaq®1-Step RT-qPCR System kit?

To conduct reverse transcription and quantitative PCR Signup and view all the answers

What is the recommended range of A260/280 readings for ensuring RNA purity?

Between 1.8 and 2.0 Signup and view all the answers

What is the purpose of the GoScript™ RT Mix in the 1-Step RT-qPCR System?

To synthesize complementary DNA from mRNA Signup and view all the answers

What is the purpose of c-MYC gene expression analysis in clinical diagnosis?

To predict the prognosis and diagnosis of aggressive B-cell lymphomas Signup and view all the answers

What is the concentration of the forward primer in the reaction mixture?

10 μM Signup and view all the answers

How many cycles of denaturation are performed in the real-time PCR reaction?

40 Signup and view all the answers

What is the temperature of the extension step in the real-time PCR reaction?

72°C Signup and view all the answers

What is the purpose of the β-actin gene in the reaction?

To act as a control gene Signup and view all the answers

What is the total volume of the reaction mixture?

20 μl Signup and view all the answers

Study Notes

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

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Description

This lab quiz covers the basics of genetic disease and molecular diagnosis, focusing on Burkitt lymphoma, a type of non-Hodgkin lymphoma. Learn about the three types of Burkitt lymphoma and the genetic changes that cause them. Test your knowledge of molecular diagnosis and its applications in genetic disease.