chain termination method Flashcards
What are the key components added to the reaction mixture in the chain-termination method of DNA sequencing? (5)
Single-stranded DNA template (the DNA to be sequenced);
All four standard nucleotides (A, T, C, G);
DNA polymerase (to join nucleotides together);
Primers required for replication;
Fluorescently-labelled modified nucleotides (added to stop replication at certain points)
What happens when a modified nucleotide is added during DNA sequencing? (2)
Replication is terminated when a modified nucleotide is incorporated into the growing DNA strand.;
This creates DNA fragments of different lengths
How are the DNA fragments separated in the chain-termination method? (4)
High-resolution electrophoresis is used to separate fragments by size;
Negatively charged DNA moves toward the positive electrode;
Smaller fragments move faster;
Fragments are visualised under UV light, and the base sequence is read from the bottom of the gel upwards
Why does the genome have to be fragmented before sequencing? (3)
The genome is very large.;
Fewer errors occur with smaller fragments;
It allows the sequencing job to be divided across different times and labs
What are the steps for sequencing an entire genome using the chain-termination method? (6)
- Genome is cut into smaller fragments using restriction enzymes;
- Fragments are inserted into bacterial artificial chromosomes (BACs);
- Each BAC is inserted into bacteria;
- Bacteria divide, creating colonies with specific DNA fragments;
- DNA is extracted, cut, and sequenced using the chain-termination method;
- Sequences are put in order to generate the sequence of the entire genome
What is next-generation sequencing and how does it differ from the chain-termination method? (2)
Next-generation sequencing (high-throughput sequencing) is much faster and automated compared to the original chain-termination method;
It allows entire genomes to be sequenced more rapidly, such as through high-throughput pyrosequencing
