DNA Hybridisation Flashcards
Which 2 processes does DNA hybridisation utilise?
- Denaturing
- Reannealing
What is the main type of DNA hybridisation?
Southern blotting
What is northern blotting?
Uses DNA to detect RNA
What is southern blotting and what does it analyse?
- Uses DNA probes to identify complementary DNA sequences after gel electrophoresis
- Analyses restriction fragments of DNA
What is the first step of southern blotting?
Digestion of genomic DNA with restriction enzymes
What happens when single stranded DNA fragments are transferred to nylon in southern blotting?
The nylon is hybridised with labelled gene probes which reform double stranded DNA
Name 3 things southern blotting can Investigate?
- Gene structure
- Gene expansion and triplet repeats
- Mutations in genetic tests
- Variation and genetic relationship
How are the results of southern blotting interpreted?
Fragments are determined by where and which sites are cut by restriction enzymes
Name a characteristic of DNA probes in blotting
- Do not have 100% similarity to the target sequence
- Do not have to completely align with the target sequence
- Do not affect the position of the target sequence on a gel
What does Sanger DNA sequence allow?
To work out the nucleotide sequence of a piece of DNA
Which nucleotides are used to achieve DNA sequencing?
ddNTPs
How are ddNTPs different to dNTPs in DNA sequencing?
- H at 3’ instead of OH
- Can be used by DNA polymerase as a substrate
- Blocks further elongation
What does DNA sequencing produce in terms of DNA fragments?
DNA molecules of different lengths depending on the position of ddNTPs
How is DNA separated and expressed in DNA sequencing?
4 separate tubes used each with a unique ddNTP and a labelled primer to initiate DNA synthesis
