3.4 DNA sequencing methods

DNA sequencing methods have revolutionized our understanding of genetics. From Sanger sequencing to next-generation techniques, scientists can now decode entire genomes rapidly. These methods allow us to read the genetic blueprint of organisms, unlocking insights into evolution, disease, and biological functions.

Advanced sequencing technologies like nanopore sequencing are pushing the boundaries further. Whole genome sequencing applications are transforming fields like personalized medicine, cancer research, and agriculture. As sequencing becomes faster and cheaper, its impact on science and society continues to grow.

Sanger Sequencing

Chain Termination Method

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• Sanger sequencing uses chain termination method to determine the nucleotide sequence of DNA
• Involves using dideoxynucleotides (ddNTPs) which lack a 3' hydroxyl group and cannot form a phosphodiester bond with the next nucleotide, terminating the DNA strand
• Four separate reactions are set up, each containing all four standard deoxynucleotides (dATP, dGTP, dCTP, dTTP) and one of the four ddNTPs (ddATP, ddGTP, ddCTP, ddTTP)
• The ddNTPs are labeled with different fluorescent dyes for detection
• The DNA fragments are then separated by size using capillary electrophoresis and the fluorescent signals are detected to determine the sequence

Shotgun Sequencing Approach

• Shotgun sequencing involves randomly breaking up the DNA into many small fragments and sequencing each fragment individually
• The fragments are cloned into vectors and transformed into bacteria to amplify the DNA
• The fragments are then sequenced using Sanger sequencing and the sequences are assembled by identifying overlapping regions to reconstruct the original DNA sequence
• Allows for sequencing of longer DNA molecules and genomes by dividing them into manageable pieces
• Overcomes the limitations of Sanger sequencing which can only sequence relatively short fragments (up to ~1000 base pairs)

Next-Generation Sequencing

High-Throughput Parallel Sequencing

• Next-generation sequencing (NGS) technologies allow for massive parallel sequencing of millions of DNA fragments simultaneously
• Enables sequencing of entire genomes much faster and cheaper than Sanger sequencing
• Most NGS methods involve fragmenting the DNA, attaching adapters to the ends, and immobilizing the fragments on a solid surface or bead
• The fragments are then amplified to form clusters and sequenced in parallel using various detection methods
• Examples of NGS platforms include Illumina, Ion Torrent, and 454 pyrosequencing

Illumina Sequencing by Synthesis

• Illumina sequencing uses a sequencing by synthesis approach with reversible dye-terminators
• DNA fragments are attached to a glass slide and amplified to form clusters
• Four fluorescently labeled reversible terminator nucleotides are added and the complementary strand is synthesized one nucleotide at a time
• After each round of synthesis, the clusters are excited by a laser and a fluorescent signal is emitted and detected to identify the incorporated nucleotide
• The terminators and fluorescent labels are then cleaved allowing the next nucleotide to be added
• Can generate up to 6 billion reads per run with read lengths up to 300 base pairs

Ion Semiconductor Sequencing

• Ion Torrent sequencing uses semiconductor technology to detect hydrogen ions released during DNA polymerization
• DNA fragments are attached to beads and clonally amplified by emulsion PCR
• The beads are then loaded onto a chip containing millions of wells, each holding a single bead
• The chip acts as a pH meter to detect changes in pH when a nucleotide is incorporated and hydrogen ions are released
• Allows for rapid sequencing without the need for expensive optical equipment
• Produces up to 60-80 million reads per run with read lengths of 200-400 base pairs

Pyrosequencing

• Pyrosequencing is a sequencing by synthesis method that detects the release of pyrophosphate during nucleotide incorporation
• DNA fragments are attached to beads and amplified by emulsion PCR
• The beads are loaded onto a PicoTiterPlate containing wells that fit a single bead
• Nucleotides are added sequentially and if a nucleotide is incorporated, pyrophosphate is released and converted to ATP by ATP sulfurylase
• The ATP drives the conversion of luciferin to oxyluciferin by luciferase, generating a light signal proportional to the number of nucleotides incorporated
• The unincorporated nucleotides are degraded by apyrase before the next nucleotide is added
• 454 pyrosequencing can produce read lengths up to 700 base pairs

Advanced Sequencing Methods

Nanopore Sequencing

• Nanopore sequencing uses protein or solid-state nanopores to directly sequence single DNA molecules without the need for amplification or labeling
• A voltage is applied across the nanopore creating an ionic current
• As a DNA strand passes through the pore, each nucleotide disrupts the current differently allowing the sequence to be determined in real-time
• Advantages include long read lengths (up to 2 megabases), portability, and ability to detect modified bases
• Limitations include high error rates and lower throughput compared to other NGS methods
• Example of nanopore sequencing platform is Oxford Nanopore Technologies' MinION device which is the size of a USB stick

Whole Genome Sequencing Applications

• Whole genome sequencing (WGS) involves sequencing the entire genome of an organism, including coding and non-coding regions
• Enables comprehensive analysis of genetic variations, structural variations, and complex traits
• Applications of WGS include:
  • Personalized medicine: identifying disease-associated variants and guiding treatment decisions based on an individual's genome
  • Cancer genomics: identifying somatic mutations and understanding tumor evolution and heterogeneity
  • Microbial genomics: studying pathogen evolution, antibiotic resistance, and disease outbreaks
  • Agricultural genomics: identifying traits associated with crop yield, quality, and stress resistance to guide breeding efforts
• Challenges of WGS include data storage and analysis, interpretation of variants of unknown significance, and ethical considerations such as privacy and informed consent