DNA Sequencing Using Enzymatic Extension Reaction

Key Concepts

• Deoxyribonucleotides vs. Dideoxyribonucleotides
  • Deoxyribonucleotides
    • Contains a hydroxyl group at position 3 of the sugar ring.
    • DNA polymerase can join the phosphate of the next nucleotide to this hydroxyl group.
  • Dideoxyribonucleotides
    • Lacks a hydroxyl group at position 3.
    • Can be added to a growing DNA chain but stops further nucleotide addition.
    • Used to terminate DNA synthesis.

DNA Sequencing Process

1. Preparation

   • DNA template is denatured in a tube.
   • Reaction mix includes:
     • Complementary primer
     • DNA polymerase
     • Four deoxyribonucleotides
     • One labeled dideoxyribonucleotide (e.g., dideoxy-A)
   • Dideoxy-A is at a lower concentration than deoxy-A.

2. Synthesis Initiation

   • Primer base pairs with the template sequence, initiating DNA synthesis.
   • DNA polymerase synthesizes DNA until a dideoxyribonucleotide is inserted.

3. Termination of Synthesis

   • Insertion of a dideoxyribonucleotide halts synthesis of that DNA molecule.
   • Optimized concentrations allow synthesis to stop at each occurrence of the base A in the new sequence.

4. Separation and Visualization

   • Fragments are separated by size using gel electrophoresis.
   • Tags on dideoxyribonucleotides enable visualization of fragments.

Comprehensive Sequencing Approach

• Reactions with Four Dideoxyribonucleotides
  • Separate reactions are conducted for each of the four bases (A, T, C, G).
  • If dideoxyribonucleotides have unique fluorescent labels, all reactions can be combined in one tube.
  • Separation results in a sequence of labeled fragments, each corresponding to a base along the DNA template.

Outcome

• The sequencing process results in fragments that provide a complete representation of the template DNA, with each base marked distinctly by different fluorescent tags.