Questions: DNA Sequencing Technologies

Question 1

What is the fundamental principle that allows Sanger sequencing to determine DNA sequence?

• A. Hybridization of labeled probes to the target sequence
• B. Random fragmentation followed by mass spectrometry
• C. Chain termination by dideoxynucleotides producing fragments of every possible length
• D. Repeated cycles of denaturation, annealing, and extension

Answer: (C)

Sanger sequencing works by including dideoxynucleotides (ddNTPs) in the synthesis reaction. When a ddNTP is incorporated instead of a normal dNTP, the chain terminates because ddNTPs lack the 3'-OH group needed for the next phosphodiester bond. By running the reaction with all four ddNTPs (each labeled with a different fluorescent dye), fragments terminating at every position are produced. Separating these fragments by size (capillary electrophoresis) and reading the fluorescent labels from smallest to largest gives the sequence.

Question 2

Illumina sequencing generates reads that are typically 10,000-50,000 base pairs long.

• T. True
• F. False

Answer: False

Illumina reads are short, typically 75-300 base pairs depending on the platform and protocol. The strength of Illumina sequencing is not read length but massive parallelism — generating hundreds of millions to billions of reads simultaneously, producing terabases of data per run at very low cost per base. Long reads (10,000+ bp) are the domain of third-generation platforms like PacBio and Oxford Nanopore, which are covered in the long-read sequencing topic.

Question 3

Explain why the transition from Sanger to next-generation sequencing enabled whole-genome sequencing to become routine.

Model answer: Sanger sequencing processes one fragment at a time through capillary electrophoresis, limiting throughput to roughly 1,000 reads per instrument per day. Sequencing a human genome required years and billions of dollars. NGS platforms parallelize the process by performing millions of sequencing reactions simultaneously on a flow cell surface, increasing throughput by orders of magnitude while reducing per-base cost by roughly a million-fold. A human genome that took 13 years and $3 billion with Sanger can now be sequenced in days for under $1,000.

The key innovation was not a fundamentally different chemistry but massive parallelization. Illumina's sequencing-by-synthesis still relies on polymerase-mediated nucleotide incorporation and fluorescent detection, but it does so for billions of clusters simultaneously rather than one capillary at a time. This throughput revolution enabled routine genome sequencing, transcriptomics, epigenomics, and metagenomics.