Practicals - Copy Number Variation (C4 genes)

Question 1

What is positional cloning?

Answer 1

Identifying the underlying genetic causes of disease using a process based on finding the location of the defective gene

Question 2

Where are complement C4 genes located?

Answer 2

In the major histocompatibility complex on human chromosome 6

Question 3

How many C4 genes can an individual have?

Answer 3

Two or up to six C4 genes

Question 4

What method can be used to determine the number of copies of the C4 gene?

Answer 4

RFLP by determining the inheritance of RFLP patterns through the family via segregation analysis

Question 5

What are the steps in C4 analysis?

Answer 5

1. extract pure DNA from B lymphoblastoid cell lines
2. DNA used in Southern hybridisation to detect RFLPs
3. Extracted DNA quantified via spectrometry
4. Sample run on Agarose gel electrophoresis (to check integrity of gDNA)
5. Complement C4 polymorphisms detected using Taq1 RFLP, restriction fragments analysed on gel and then used for southern analysis
6. C4 RFLP blots hybridised with C4-specific probes
7. Probe binds to complementary DNA
8. Viewed using autoradiography

Question 6

What is the best method to determine CNVs?

Answer 6

Carefully designed genomic RFLP analysis with appropriate probes for hybridization

Question 7

What happens in real-time PCR?

Answer 7

A fluorescent dye is incorporated into the amplified DNA and emits light proportional to the number of amplified copies as the PCR proceeds