DNA Sequencing Techniques

Why This Matters

DNA sequencing is the foundation of modern genetics—every major advance from disease diagnosis to evolutionary analysis depends on our ability to read genetic code. You're being tested not just on knowing these techniques exist, but on understanding why each method was developed, what trade-offs it involves, and when you'd choose one over another. The core concepts here include chain termination chemistry, sequencing by synthesis, read length versus throughput trade-offs, and computational assembly strategies.

Don't just memorize technique names and dates. Know what problem each method solves: Why do we need long reads for some projects but short reads work fine for others? Why did scientists move from chemical cleavage to enzymatic methods? When you can explain the underlying mechanisms and compare approaches, you'll nail both multiple-choice questions and FRQs that ask you to design an experiment or troubleshoot a sequencing strategy.

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Chain Termination and Chemical Methods

These foundational approaches established the principles of DNA sequencing by generating fragments of different lengths and separating them to read the sequence. The key insight: if you can control where synthesis stops, you can determine sequence position.

Sanger Sequencing (Chain Termination Method)

• Dideoxynucleotides (ddNTPs) lack a 3'-OH group—this terminates strand elongation when incorporated, producing fragments ending at every position of a given base
• Capillary electrophoresis separates fragments by size—fluorescent labels on each ddNTP type allow automated detection and sequence readout
• Gold standard for accuracy (up to ~1000 bp)—still used to validate NGS results and sequence individual genes or plasmids

Maxam-Gilbert Sequencing

• Chemical cleavage method—uses specific reagents to break DNA at G, A, T, or C residues, generating base-specific fragment ladders
• Requires radioactive labeling—fragments are visualized by autoradiography after gel electrophoresis, making it labor-intensive and hazardous
• Historically significant but largely obsolete—replaced by Sanger sequencing due to complexity and safety concerns

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Short-Read Next-Generation Sequencing

NGS platforms achieve massively parallel sequencing—millions of fragments sequenced simultaneously. The trade-off: shorter individual reads but enormous throughput and dramatically lower cost per base.

Illumina Sequencing

• Sequencing by synthesis with reversible dye terminators—each nucleotide addition is detected optically, then the terminator is cleaved to allow the next incorporation
• Short reads (50-300 bp) but extremely high throughput—billions of reads per run make it ideal for whole-genome sequencing, RNA-seq, and population studies
• Dominant platform in genomics research—cost-effective and highly accurate, though short reads complicate assembly of repetitive regions

Ion Torrent Sequencing

• Semiconductor detection of pH changes—when a nucleotide is incorporated, a proton is released; the resulting pH shift is measured electronically (no optics required)
• Faster run times and lower instrument cost—produces reads up to ~400 bp, suitable for targeted panels and smaller projects
• Struggles with homopolymer regions—multiple identical bases in a row cause signal interpretation errors, limiting accuracy in some contexts

Pyrosequencing

• Detects pyrophosphate (PPi) release during synthesis—PPi triggers a luciferase reaction producing light proportional to nucleotides incorporated
• Real-time detection enables quantitative applications—useful for SNP genotyping and methylation analysis where you need to measure allele frequencies
• Limited read length (~300 bp)—largely superseded by Illumina for most applications but still valuable for targeted quantitative assays

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Long-Read Sequencing Technologies

Long reads solve the assembly problem: repetitive regions, structural variants, and complex genomes require reads that span entire repeat units. The mechanism shift: observe single molecules directly rather than amplified clusters.

Single-Molecule Real-Time (SMRT) Sequencing

• Observes DNA polymerase incorporating nucleotides in real time—each base has a distinct fluorescent label detected in zero-mode waveguides (no amplification bias)
• Ultra-long reads (10,000-30,000+ bp)—enables phasing of haplotypes and resolution of complex structural variants
• Higher error rate per read but correctable—circular consensus sequencing (multiple passes) achieves high accuracy; ideal for de novo assembly

Nanopore Sequencing

• Measures ionic current changes as DNA translocates through a protein pore—each base causes a characteristic current disruption, enabling direct sequence readout
• Reads can exceed 1 million bp—no theoretical upper limit, making it uniquely suited for spanning entire repetitive regions or even full chromosomes
• Portable and requires minimal sample prep—the MinION device enables field-based sequencing for outbreak surveillance and environmental metagenomics

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Sample Preparation and Assembly Strategies

These approaches aren't sequencing chemistries themselves but are essential for generating and interpreting sequence data. Understanding when to use each is critical for experimental design questions.

Polymerase Chain Reaction (PCR)

• Exponential amplification through thermal cycling—denaturation ($\sim$95°C), primer annealing ($\sim$55°C), and extension ($\sim$72°C) repeated 25-35 cycles
• Essential upstream step for most sequencing workflows—generates sufficient template from minute samples; also used for targeted enrichment
• Introduces amplification bias—GC-rich or repetitive regions may amplify unevenly, which is why amplification-free methods (SMRT, Nanopore) are valuable

Shotgun Sequencing

• Random fragmentation followed by sequencing and computational assembly—overlapping reads are aligned to reconstruct the original sequence
• Enabled the Human Genome Project—combined with Sanger sequencing initially, now paired with NGS for efficient whole-genome assembly
• Assembly quality depends on coverage and read length—short reads require higher coverage; long reads simplify assembly of repetitive regions

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Quick Reference Table

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Self-Check Questions

1. Which two sequencing methods produce long reads without requiring PCR amplification, and what detection mechanism does each use?

2. Compare Sanger sequencing and Illumina sequencing: What do they share in terms of basic chemistry, and how do they differ in throughput and read length?

3. A researcher needs to sequence a bacterial genome with many repetitive transposon insertions. Which sequencing platform would you recommend and why?

4. What is the key limitation shared by Ion Torrent and Pyrosequencing when sequencing homopolymer regions, and what causes this problem?

5. If you needed to detect DNA methylation patterns without bisulfite conversion, which sequencing approach would preserve this epigenetic information, and what feature of the technology makes this possible?