MCGB - DNA Hybridisation

Question 1

What is meant by the term ssDNA?

Answer 1

Single stranded DNA

Question 2

Outline the stages of Southern blotting.

Answer 2

• digest genomic DNA with restriction enzymes
• separate DNA fragments by gel electrophoresis
• transfer DNA fragments to nylon
• hybridise filter with labelled gene probe
• detect hybridisation by exposure of filter to x-ray film

Question 3

What two ways is DNA transferred to the nylon/nitrocellulose filter in a Southern blot?

Answer 3

Blotted by capillary action using paper towels, or using electrophoretic transfer (a voltage is applied across the set-up so that negatively charged DNA moves from the gel onto the filter).

Question 4

Is the DNA on the filter in single or double stranded form?

Answer 4

Single-stranded

Question 5

Give some reasons for using Southern hybridisation.

Answer 5

• to investigate gene structure
• to investigate gene expansions/triplet repeats
• to investigate mutations in genetic tests
• to investigate variation/genetic relationships

Question 6

Give some limitations of DNA probes in Southern blotting

Answer 6

• probes do not have 100% similarity to target sequence
• probes do not completely align with target sequence
• probes do not affect position of target sequence on a gel

Question 7

What is the Sanger chain termination method?

Answer 7

A method of DNA sequencing that uses fluorescent deoxynucleotides.