DNA Sequencing
Application: Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing
- DNA sequencing allows for the nucleotide base sequence of an organism's genetic material to be determined
- Most methods for sequencing DNA involve the use of chain-terminating dideoxynucleotides
- The dideoxy chain-termination method was developed by Frederick Sanger in 1977
- The chain-termination method uses modified nucleotides called dideoxynucleotides
- Dideoxynucleotides have a slightly different structure to standard nucleotides
- They lack the 3’-hydroxyl group so cannot form a covalent bond with the next nucleotide to be incorporated by DNA polymerase
- Dideoxynucleotides prevent elongation of the nucleotide chain, which therefore terminates
- Advances in technology have enabled the development of rapid high-throughput sequencing methods which allow scientists to sequence the genomes of organisms rapidly
Once the dideoxynucleotide is added to the developing strand DNA polymerase stops the replication of the developing DNA strand to produce a shortened DNA chain
The chain termination method in action
- DNA sample of interest is used as a template in chain-termination PCR
- Deoxynucleotides and fluorescently-labelled dideoxynucleotides are used
- In the extension step of PCR, DNA polymerase will incorporate deoxynucleotides
- If a dideoxynucleotide is randomly incorporated, extension stops
- Because of the nature of PCR, billions of copies of the DNA sequence of interest will be produced that will be terminated (by a dideoxynucleotides) at random lengths
- The fragments can separated by size in gel electrophoresis
- The fluorescent marker corresponds to a particular ‘terminator’ nucleotide and can be visualised
- This allows the base sequence to be built up one base at a time
High-throughput method of carrying out the chain termination method