DNA sequencing Flashcards
what is DNA sequencing
the process of determining the precise order of nucleotides within a DNA molecule
What is Dideoxy chain termination (Sanger sequencing) and what are its characteristics
DNA sequencing technique
- low error rate and highly reliable
- ‘gold standard’ technique meaning other sequencing techniques are compared to it
What is Automated DNA Sequencing by ABI 3730
Can perform sequencing of multiple samples simultaneously, with a very high accuracy
Disadvantage of automated DNA sequencing
only performs the separation of labelled DNA and determined the sequences, however the whole sequencing process still requires considerable hands preparation prior to sequencing
What are the Basic Dideoxy chain termination steps
1) Single stranded DNA template generated
- done by DNA denaturation
2) Enzymatic Sequencing Reaction
- DNA polymerase makes multiple copies of the DNA
3) Size Separation of Products by Electrophoresis
- labelled DNA molecules separated by size
4) Detection of Reaction Products
- sequential detection of the terminating nucleotide to identify base
5) Readout of Sequence
- reconstructing the sequence
Requirements of Dideoxy chain termination
4 reaction mixtures set up including:
Primer needed to anneal/hybridise to template strand
DNA Polymerase recognises this partially dsDNA molecule and forms initiation complex
Elongates this using dNTPs from the 3’OH terminus of the primer in a 5’ to 3’ direction. Ester bond formed between free 3’OH group and phosphate on dNTP. Mg2+ required as a cofactor.
One kind of ddNTP (labelled with different fluorescent molecule) added to each of the 4 reaction mixtures to terminate the reaction, at a low molar ratio compared to the dNTPs. If a ddNTP is incorporated into the strand during elongation, elongation is terminated because there is no free 3’OH group on ddNTP to react with phosphate of dNTP, and this prevents further extension. Polymerase then dissociates and strand is released from enzyme.
