DNA Sequencing

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8 Terms

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What is DNA Sequencing

-used to read the order of the bases (A,T,C,G) in DNA

-helps us understand genetic traits, mutations, and diseases

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Why is it important?

Important for:

  • Diagnosing genetic conditions

  • Identifying organisms (forensics, ancestry)

  • Studying gene function

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What is First Generation Sequencing?

Includes Maxam-Gilbert and Sanger Sequencing

  • Maxam-Gilbert: Involves chemical reactions to break DNA at specific bases. Rarely used today (complex and hazardous).

  • Sanger Sequencing (dideoxy method): Uses chain-terminating nucleotides (ddNTPs) to determine DNA sequence. Very accurate, often called the "gold standard."

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Second Generation Sequencing (Next-Generation Sequencing or NGS)

  • Faster and can sequence millions of DNA fragments at once (massively parallel)

  • Uses fluorescent tags and image-based detection

  • Popular methods: Illumina, Ion Torrent

  • Common in clinical testing and research labs

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Third Generation Sequencing

  • Sequences single DNA molecules in real time

  • Doesn’t require PCR amplification

  • Produces longer reads (can span entire genes)

  • Examples: PacBio, Oxford Nanopore

  • Useful for detecting structural variations and whole-genome sequencing

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Sanger Sequencing (Dideoxy Method)

  • Based on DNA replication

  • Uses ddNTPs (dideoxynucleotides)

    • ddNTPs lack a 3'-OH group → stops replication when added

  • DNA polymerase can’t add another base after a ddNTP → creates fragments of different lengths

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Steps in Sanger Sequencing:

  1. Amplify DNA (using PCR or cloning)

  2. Denature DNA → becomes single-stranded

  3. Add to reaction tube:

    • Primer

    • DNA polymerase

    • 4 dNTPs (normal bases)

    • 4 ddNTPs (labeled with fluorescent dyes or radioactive markers)

  4. DNA synthesis begins, but stops whenever a ddNTP is added

  5. Fragments are separated by gel electrophoresis

  6. Fluorescence or radioactive signal tells you the last base in the strand

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Key Terms

  • ddNTP: Dideoxynucleotide (chain terminator)

  • dNTP: Normal DNA building block

  • Primer: Short DNA that starts the copying process

  • Electrophoresis: Method for separating DNA pieces by size

  • Fluorescent label: Helps visualize the sequence