DNA sequencing refers to the process by which the base order of a nucleotide sequence is elucidated
Dideoxynucleotides
- The most widely used method for DNA sequencing involves the use of chain-terminating dideoxynucleotides
Dideoxynucleotides
- Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group necessary for forming a phosphodiester bond
- Consequently, ddNTPs prevent further elongation of a nucleotide chain and effectively terminate replication
- The resulting length of a DNA sequence will reflect the specific nucleotide position at which the ddNTP was incorporated
- For example, if a ddGTP terminates a sequence after 8 nucleotides, then the 8th nucleotide in the sequence is a cytosine
Determining Nucleotide Positions Using Dideoxynucleotides
Sequencing
Dideoxynucleotides can be used to determine DNA sequence using the Sanger method
Dideoxynucleotides can be used to determine DNA sequence using the Sanger method
- Four PCR mixes are set up, each containing stocks of normal nucleotides plus one dideoxynucleotide (ddA, ddT, ddC or ddG)
- As a typical PCR will generate over 1 billion DNA molecules, each PCR mix should generate all the possible terminating fragments for that particular base
- When the fragments are separated using gel electrophoresis, the base sequence can be determined by ordering fragments according to length
- If a distinct radioactive or fluorescently labelled primer is included in each mix, the fragments can be detected by automated sequencing machines
- If the Sanger method is conducted on the coding strand (non-template strand), the resulting sequence elucidated will be identical to the template strand
DNA Sequencing via the Sanger Method