DNA Sequencing: Sanger Method Overview

DNA Sequencing

• DNA sequencing determines the order of bases in a DNA sequence.
• It has aided in our understanding of genetics, with applications such as tackling human diseases and studying evolution.

Sanger Sequencing Method

• Also known as the "chain termination method."
• A first-generation sequencing method.
• Developed in 1977 by Frederick Sanger and his colleagues (who later sequenced an entire virus genome).
• Uses high fidelity DNA polymerase to create a complementary copy from a single-stranded DNA template.
• Requires a single primer complementary to the template to initiate DNA synthesis from the 3' end.

Sanger Sequencing: Chain Termination Method

• Uses ddNTPs (dideoxynucleotides) which resemble DNA monomers and are incorporated into the growing chain.
• ddNTPs differ from dNTPs in two ways:
  • They lack a 3' hydroxyl group needed for further DNA extension, causing chain termination upon incorporation.
  • Each ddNTP is tagged with a fluorescent dye, allowing for automatic detection.

Components for Sanger Sequencing

• DNA template
• Primer
• dNTPs (dATP, dTTP, dCTP, dGTP)
• ddNTPs (ddATP, ddTTP, ddCTP, ddGTP)
• DNA polymerase

Sanger Sequencing Method: Steps

• Step 1: The DNA to be sequenced is amplified using PCR, and the double helix is denatured with heat to separate the strands.
• Step 2: A mixture of dNTPs (normal nucleotides), ddNTPs (labeled dideoxynucleotides), DNA primer, and DNA polymerase is added to the template.
• Step 3: The polymerase creates a copy of the DNA template, but the process is terminated when a ddNTP is incorporated, resulting in fragments of varying lengths.
• Step 4: Fragments are separated by capillary electrophoresis, where shorter fragments move faster due to their smaller size. The laser excites the fluorescent dye on each ddNTP, emitting light for detection. This creates a chromatogram that shows the fluorescent peaks of each nucleotide, indicating the sequence from shortest to longest.

Human Genome Project

• The first major effort in DNA sequencing was the Human Genome Project.
• This project used Sanger Sequencing (chain termination method)
• The project took 13 years to complete.
• The project cost $3 billion.
• The project was completed in 2003.

Next Generation Sequencing (NGS)

• NGS is a newer technology.
• Second-generation sequencing was introduced in 2004 and 2006.
• Offers high-throughput sequencing.

Applications of NGS

• NGS can be used to sequence the entire genome.
• NGS can be used to sequence specific regions of DNA or RNA.
• NGS has revolutionized the life sciences.
• Used to discover novel RNA variants and splice sites.
• Used to quantify mRNAs for gene expression analysis.
• Used to study epigenetic factors.
• Used to study cancer samples by studying rare somatic variants and tumor subclones.
• Used to identify novel pathogens.

Categories of NGS

• Sequencing by hybridization: uses specific probes to identify sequences.
  • Used for diagnostic purposes, including:
    • identifying disease-related SNPs.
    • identifying gross chromosome abnormalities, such as rearrangements, deletions, duplications, and copy number variants (CNVs).
• Sequencing by Synthesis (SBS): an improvement on Sanger Sequencing that does not use ddNTPs.
  • Uses repeated synthesis cycles and methods to incorporate nucleotides into a growing chain.
  • There are other methods like Pyrosequencing but are not discussed here.

Similarities Between Different NGS Technologies

• Sample preparation:
  • Requires a library obtained by amplification or ligation with custom adapter sequences.
  • Adapter sequences allow library hybridization to the sequencing chips.
    • These sequences provide a universal priming site for sequencing primers.
• Sequencing Machines:
  • Each library fragment is amplified on a solid surface – either beads or a flat silicon-derived surface.
  • This is done using covalently attached DNA linkers, which hybridize the library adapters.
  • Amplification creates clusters of DNA, each originating from a single library fragment.
    • Each cluster acts as an individual sequencing reaction.
    • The sequence from each cluster is optically read, either by light or fluorescence.
    • Each machine has its cycle conditions.
• Data Output:
  • Each machine provides raw data at the end of the sequencing run.
  • Raw data is a collection of DNA sequences generated at each cluster.
  • This data is further analyzed to provide meaningful results.

Sequencing by Synthesis (SBS) Properties

• Relies on shorter reads (300-500 bp).
• Generally, it has a higher error rate than Sanger sequencing.
  • Due to incomplete removal of the fluorescent signal, which can cause higher background noise levels.
• Relies on high sequence coverage, which is referred to as “massively parallel sequencing”.
  • Of millions to billions of short DNA sequence reads (50 – 300 nucleotides)
  • This is called Short Read Sequencing.
• Utilizes step-by-step incorporation of reversibly fluorescent and terminated nucleotides.
  • Nucleotides are modified in two ways:
    • Each nucleotide is reversibly attached to a single fluorescent molecule with unique emission wavelengths.
    • Each nucleotide is reversibly terminated, ensuring only one nucleotide is incorporated per cycle.

Sequencing by Synthesis (SBS) Process

• All four nucleotides are added to the sequencing chip.
• A single nucleotide is incorporated into the sequence.
• Remaining nucleotides are washed away.
• The fluorescent signal is read at each cluster and recorded.
• Both the fluorescent molecule and the terminator group are cleaved and washed away.
• This process is repeated until sequencing is complete.

Illumina Sequencing (Solexa Sequencing) - Cyclic Reversible Termination

• Step 1: Library Preparation

  • DNA is broken up into more manageable fragments (~200 – 600 bp).
  • Short sequences of DNA (adapters) are attached to the DNA fragments.
  • The DNA fragments attached to the adapters are denatured (made single-stranded).
  • This process constructs a mix of adapter-flanked fragments up to several hundred bp in length.

• Step 2: Bridge Amplification

  • Illumina technology relies on bridge PCR to amplify the genomic region being sequenced.
  • An in vitro constructed adapter-flanked library is PCR amplified.
    • Both primers densely coat the surface of a solid substrate, attached at their 5’ end by a flexible linker.
    • Due to this, amplification products from the library remain locally attached near the point of origin.

• Step 3: Sequencing Reaction

  • Components:

    • Primers
    • dNTPs: labelled with a fluorescent dye and contain a reversible terminator (Trinitrogen = N3)
    • DNA polymerase

  • Process:

    • Cyclic reversible termination
    • Only one of four fluorescent dNTPs is added per cycle.
    • Images of clusters are captured after the incorporation of each nucleotide.
    • After imaging, the fluorescent dye and terminator are cleaved and released.

When to Use NGS vs Sanger Sequencing

• Sanger Sequencing:
  • A good choice when investigating a small region of DNA on a limited number of samples or genomic targets (~20 or fewer).
• NGS:
  • Allows for cost-effective screening of more samples.
  • Allows for the detection of multiple variants across targeted areas of the genome.
  • Used when Sanger sequencing is too costly and time-consuming.