MCGB - DNA Hybridisation Flashcards
1
Q
What is meant by the term ssDNA?
A
Single stranded DNA
2
Q
Outline the stages of Southern blotting.
A
- 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
3
Q
What two ways is DNA transferred to the nylon/nitrocellulose filter in a Southern blot?
A
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).
4
Q
Is the DNA on the filter in single or double stranded form?
A
Single-stranded
5
Q
Give some reasons for using Southern hybridisation.
A
- to investigate gene structure
- to investigate gene expansions/triplet repeats
- to investigate mutations in genetic tests
- to investigate variation/genetic relationships
6
Q
Give some limitations of DNA probes in Southern blotting
A
- 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
7
Q
What is the Sanger chain termination method?
A
A method of DNA sequencing that uses fluorescent deoxynucleotides.