DNA sequencing | Khan Academy

Steps of DNA Sequencing

Step 1: DNA Amplification

  • Start with a sample of DNA.

  • Use Polymerase Chain Reaction (PCR) to amplify the DNA sample.

  • PCR generates numerous DNA fragments.

Step 2: Incorporating Nucleotides

  • Add normal deoxynucleotides for DNA strand growth.

  • A normal nucleotide structure includes:

    • OH group

    • H group

    • Base

    • Carbon group and oxygen-hydrogen combination

  • Introduce dideoxynucleotides (ddNTPs):

    • Structurally similar to regular nucleotides except missing one oxygen (only has H)

    • Incorporating a ddNTP terminates strand elongation, preventing further growth.

Fluorescent Labeling of dideoxynucleotides

  • Fluorescently label ddNTPs:

    • G's: Blue

    • A's: Red

    • T's: Green

    • C's: Orange

  • As ddNTPs are incorporated during PCR, multiple DNA fragments of various lengths are generated.

Example of Elongation Termination

  • A growing strand incorporates:

    • A regular nucleotide, then another, and then a dideoxynucleotide (stopping elongation).

    • Resulting fragment length varies based on incorporation points of ddNTPs.

Step 3: Gel Electrophoresis

  • Separate DNA fragments by size using gel electrophoresis.

  • Fragments migrate through gel; smaller strands move faster than longer ones.

  • A computer analyzes the fluorescent labels to determine the sequence:

    • Reads the color signals and deduces the corresponding nucleotides (e.g., blue for G, red for A).

Final Outcome

  • The resulting DNA sequence is determined by analyzing the order of fluorescent labels.