DNA Sequencing Overview

What is DNA Sequencing

  • Lab technique to determine the sequence of bases (A, C, G, T) in DNA.
  • Provides genetic information from DNA segments, genomes, or microbiomes.
  • Used for:
    • Screening features of genes.
    • Evolutionary analysis among species and populations.
    • Diagnosing and treating diseases.
    • Epidemiological studies.

Sanger Sequencing

  • Developed in 1977 by Frederick Sanger.
  • Analyzes DNA segments typically less than 1,000 base pairs (bp) in length.
Requirements:
  1. Template DNA
  2. DNA primer complementary to the template.
  3. Four deoxynucleotide triphosphates (dNTPs).
  4. DNA polymerase to extend the primer by adding dNTPs.
  5. Four dideoxynucleotide triphosphates (ddNTPs) labeled with fluorescent dye which terminates synthesis.
    • ddNTPs differ from dNTPs by missing one oxygen atom, preventing them from linking with the next nucleotide.
Stages of Sanger Sequencing:
  1. Denature double-stranded DNA into single-stranded DNA; insert into plasmids and bacterial cells for multiplication.
  2. Attach primer.
  3. Prepare four polymerase solutions with dNTPs, each containing one specific ddNTP.
  4. Initiate DNA synthesis; chain extends until termination nucleotide is added.
  5. Denature resulting fragments into single-stranded DNA.
  6. Separate fragments using capillary gel electrophoresis to determine sequence.

Next Generation Sequencing (NGS)

  • Revolutionizes genomic research by allowing simultaneous sequencing of millions of fragments.
  • Polymerase incorporates fluorescent nucleotides one by one onto the template strand, initiated by a primer.
  • Identifying incorporated nucleotides via fluorescent tags.
  • Capable of sequencing hundreds of thousands of genes at once, enhancing the detection of novel variants.