Introduction to Sanger sequencing
Sanger sequencing, developed by Frederick Sanger and colleagues in 1977, is a pioneering method for DNA sequencing. Sanger sequencing involves the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication and electrophoresis and. Initially, it was the predominant sequencing method for four decades, first commercialized by Applied Biosystems in 1986. Key highlights:
- The first direct DNA sequencing method
- Sequencing by synthesis
- Di-deoxy termination method
- DNA polymerase + Pool of unmodified dNTPs + Pool of di-deoxy NTPs
Methodology of Sanger sequencing
It uses the chain-termination method which requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotide triphosphates (dNTPs), and di-deoxynucleotide triphosphates (ddNTPs), lacks the 3′ hydroxyl group on the deoxyribose sugar which terminate DNA strand elongation causing DNA polymerase to stop the extension modified ddNTP is incorporated. The modified ddNTPs may be radioactively (manual detection method) or fluorescently labeled for detection in automated sequencing machines.
What is the difference between a deoxyribonucleotide and a dideoxyribonucleotide? Why dideoxyribonucleotide is used in Sanger's method of DNA sequencing? What will happen if very high or very low amount of ddNTPs are used in Sanger's method of DNA sequencing? Discuss.Difference between a deoxyribonucleotide and a dideoxyribonucleotide
The key difference between a deoxyribonucleotide and a dideoxyribonucleotide lies in their chemical structure. A deoxyribonucleotide has a deoxyribose sugar, a nitrogenous base (adenine, guanine, cytosine, or thymine), and a phosphate group. On the other hand, a dideoxyribonucleotide lacks the 3′ hydroxyl group on the deoxyribose sugar, making it incapable of forming further phosphodiester bonds.
Dideoxyribonucleotides are used in Sanger’s method of DNA sequencing as chain terminators. When a dideoxyribonucleotide is incorporated into a growing DNA strand during the sequencing reaction, it prevents further elongation of that strand, resulting in DNA fragments of varying lengths.
| Property | Deoxyribonucleotide | Dideoxyribonucleotide |
|---|---|---|
| 3′ Hydroxyl Group | Present | Absent |
| Forms Phosphodiester Bonds | Yes | No |
| Function in Sanger Sequencing | Allows strand elongation | Terminates strand elongation |
 |  |
If very high amounts of ddNTPs (dideoxyribonucleotides) are used in Sanger’s method: More DNA strands will terminate prematurely resulting in shorter fragment lengths and potentially leading to incomplete or inaccurate sequence information
If very low amounts of ddNTPs are used: Fewer DNA strands will terminate resulting in longer fragment lengths, potentially making it difficult to resolve and distinguish individual fragments. this leads to incomplete or inaccurate sequence information.
Chain-termination method for DNA sequencing
- The DNA sample is divided into four separate sequencing reactions, containing all four standard deoxynucleotides (dATP, dGTP, dCTP, and dTTP) and the DNA polymerase.
- Only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) is added to each reaction; the other nucleotides are ordinary ones.
- The deoxynucleotide concentration should be approximately 100-fold higher than that of the corresponding dideoxynucleotide (e.g. 0.5mM dTTP: 0.005mM ddTTP) to allow enough fragments to be produced while still transcribing the complete sequence
- Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat-denatured and separated by size using gel electrophoresis.
- The DNA bands can be visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image.
DNA Sequence Analysis by Autoradiograph
Polyacrylamide gel electrophoresis (PAGE) is performed after the completion of each reaction. Each reaction mix is loaded in a separate well, and it’s performed under denaturing conditions in the presence of urea or less frequent formamide, conditions must be carefully controlled to separate the strands that might differ even with just a single nucleotide. Urea and formamide lower the melting point of DNA molecules, denature DNA by disrupting the H bond and the newly synthesized strand separates from the template strand.
Electrophoresis is carried out at high voltage to prevent the renaturation of DNA due to high heat generation in the gel.
After the complete run, the gel is transferred on a nitrocellulose filter and autoradiography is performed so that only bands having the 5’ radiolabelled molecule will be visible as bands. In PAGE the shortest fragment moves faster so the bottommost molecule is the first dideoxynucleotide which stopped the chain elongation by its incorporation and thus that should be the first sequenced nucleotide.
Automated Sanger sequencing
It represents a significant advancement in DNA sequencing technology, building upon Sanger’s original chain-termination method. The process employs fluorescently labeled dideoxynucleotides (ddNTPs) and capillary electrophoresis for automated sequence determination. Key features include four-color fluorescent detection, automated sample processing with 8-96 capillaries, and computer-controlled base calling. This high-throughput method delivers read lengths of 600-900 bases with up to 99.9% accuracy, making it invaluable for mutation detection, SNP validation, pathogen identification, and NGS result verification. Its advantages of minimal manual intervention, standardized protocols, and cost-effectiveness for small-scale projects have established it as the gold standard for targeted DNA sequencing applications, particularly in clinical diagnostics and validation studies.
| Aspect | Description | Technical Details |
|---|---|---|
| Instrumentation Components | • Capillary electrophoresis system • Laser detection unit • Temperature control module • Automated sample loader • Computer interface | • Signal: noise ratio >50 • Resolution >0.4 • Peak spacing verification • Quality scores (QV20+) • Read length monitoring |
| Chemistry Components | • Fluorescent dye-labeled ddNTPs • DNA polymerase • Buffer system • Polymer matrix • Internal size standard | • BigDye™ or similar terminators • Modified Taq polymerase • POP™ polymers (POP-4, POP-6, POP-7) • ROX™-labeled size standards |
| Sample Preparation | • Template purification • Cycle sequencing reaction • Post-reaction cleanup • Sample resuspension | • 5-20 ng template DNA • 25-35 thermal cycles • Ethanol/EDTA precipitation • Hi-Di™ formamide resuspension |
| Automation Features | • Automated sample injection • Real-time data collection • Quality monitoring • Base calling • Data analysis | • Electrokinetic injection (15s @ 1-2kV) • Adaptive baseline tracking • Quality value assignment • Phred-based base calling • Fragment analysis capabilities |
| Run Parameters | • Injection conditions • Electrophoresis settings • Run temperature • Data collection • Analysis parameters | • 1-3 kV injection voltage • 8-15 kV run voltage • 50-60°C run temperature • 1-3 hour run time • Variable sampling rates |
| Quality Control | • Signal intensity monitoring • Background noise analysis • Peak resolution check • Internal standard tracking • Base quality scoring | • Signal:noise ratio >50 • Resolution >0.4 • Peak spacing verification • Quality scores (QV20+) • Read length monitoring |
| Data Output | • Raw data files • Analyzed sequences • Quality reports • Electropherograms • Text files | • .ab1/.abi files • FASTA format • Quality value files • Trace files • Analysis reports |
| Common Applications | • De novo sequencing • Mutation detection • SNP validation • Fragment analysis • Microsatellite analysis | • 600-900 bp read length • 99.9% accuracy • Resolution to single base • Size accuracy ±1 bp • Multiplexing capability |
| System Maintenance | • Capillary array replacement • Polymer block maintenance • Buffer replacement • Laser optimization • Calibration routines | • 100-150 runs per array • Weekly maintenance • Daily buffer changes • Monthly calibrations • Regular performance tests |
| Troubleshooting | • Signal intensity issues • Peak resolution problems • Base calling errors • Sample failures • System errors | • Matrix/spectral calibration • Spatial calibration • Injection troubleshooting • Sample quality checks • Error code diagnostics |
Applications of Sanger Sequencing
| Application Category | Specific Applications | References |
|---|---|---|
| Clinical Diagnostics | • Mutation detection in hereditary diseases • Cancer-associated gene variants • Pharmacogenetic testing • Confirmation of NGS findings | • Rehm et al., Nat Rev Genet 14:295-306 (2013) • Beck et al., J Mol Diagn 18:851-62 (2016) |
| Microbial Identification | • 16S rRNA sequencing for bacterial identification • Fungal ITS region analysis • Viral genome sequencing • Antimicrobial resistance genes | • Clarridge JE, Clin Microbiol Rev 17:840-62 (2004) • Schoch et al., PNAS 109:6241-6 (2012) |
| Research Applications | • Gene characterization • Molecular evolution studies • Population genetics • Phylogenetic analysis | • Yang & Rannala, Nat Rev Genet 13:303-14 (2012) • Nielsen & Slatkin, Pop Genet Theory (2013) |
| Forensic Science | • mtDNA analysis • Species identification • DNA profiling validation • Mixture analysis | • Butler JM, Forensic DNA Typing (2015) • Parson et al., FSI Genet 13:134-42 (2014) |
| Agricultural Applications | • Crop genome analysis • Livestock breeding • Plant pathogen identification • GMO verification | • Edwards & Batley, Plant Biotech J 8:2-9 (2010) • Yang et al., Nat Biotech 25:1239-45 (2007) |
| Quality Control | • Plasmid verification • Cell line authentication • Primer binding site confirmation • Cloning verification | • ATCC Standards Dev Org, Nat Rev Cancer 10:441-8 (2010) |
| Environmental Studies | • Biodiversity assessment • Environmental monitoring • Species identification • Metabarcoding validation | • Valentini et al., TREE 24:110-7 (2009) • Taberlet et al., Mol Ecol 21:2045-50 (2012) |
| Drug Development | • Target gene sequencing • Mutation screening • Vector confirmation • Cell line verification | • Metzker ML, Nat Rev Genet 11:31-46 (2010) • Lone et al., Mol Ther 17:2121-9 (2009) |
Key differences between Sanger sequencing and NGS
| Feature | Sanger Sequencing | Next-Generation Sequencing (NGS) |
| Principle | Chain-termination method | Parallel sequencing of millions of DNA fragments |
| Development | Introduced in 1977 by Frederick Sanger | Emerged in the mid-2000s |
| Read length | > 500 nucleotides (long reads) | Short reads (100s of base pairs), with some platforms achieving longer reads |
| Accuracy | Very high (around 99.99%) | High, but may vary between platforms |
| Speed | Slower | Faster, capable of high-throughput sequencing |
| Cost per Bae | Higher cost per base | Lower cost per base |
| Applications | Often used for smaller-scale projects, validation of results | Commonly used for large-scale genome analysis, whole-genome sequencing, transcriptomics, etc. |
| Instrumentation | Requires capillary electrophoresis machines | Requires specialized NGS platforms |
| Labour intensity | Labor-intensive, manual processes | Less labor-intensive due to automation |
| Error rate | Low error rate (high accuracy) | Low, but may vary by platform and read length |
| Use in public health | Active role in initiatives like sequencing the spike protein of SARS-CoV-2, surveillance of norovirus outbreaks through CDC’s CaliciNet | Commonly used in various public health initiatives due to high-throughput capabilities and rapid results |
Human Genome Sequencing Project
- Too expensive and time-consuming!
- Completed in 2003, took ~13 years
- Cost: USD 3 billion
Comparison of Sanger Sequencing and PCR
| Parameter | Sanger Sequencing | Polymerase Chain Reaction (PCR) |
|---|---|---|
| Primary Purpose | DNA sequence determination | DNA amplification |
| Basic Principle | Chain-termination method using dideoxynucleotides (ddNTPs) | Exponential amplification using thermal cycling |
| Read Length | Up to ~900-1000 base pairs | Typically <10 kb, optimally 0.1-4 kb |
| Accuracy | 99.9% accuracy for high-quality reads | Depends on polymerase (Taq ~1 error/10⁴ bases; high-fidelity enzymes ~1 error/10⁶ bases) |
| Key Components | – DNA template – DNA polymerase – Primers – dNTPs – Fluorescently labeled ddNTPs | – DNA template – Thermostable DNA polymerase – Primers – dNTPs – Mg²⁺ |
| Steps | 1. Denaturation 2. Primer annealing 3. Extension with ddNTP termination 4. Capillary electrophoresis 5. Fluorescence detection | 1. Denaturation (94-96°C) 2. Primer annealing (50-65°C) 3. Extension (72°C) 4. Repeat cycles 25-35 times |
| Applications | – Mutation detection – SNP validation – Gene identification – Pathogen identification – Validation of NGS results | – DNA amplification for analysis – Diagnostic testing – Forensic analysis – Gene expression studies – Cloning |
| Limitations | – Single DNA fragment at a time – Limited throughput – Difficulty with repetitive sequences – High background noise in mixed samples | – Limited fragment size – Potential for non-specific amplification – PCR inhibitors can affect results – Primer design crucial |
| Time Required | 2-3 hours for sequencing reaction + 2-3 hours for analysis | 1-4 hours depending on fragment size and cycles |
| Equipment Needed | Thermal cycler, automated sequencer, computer for analysis | Thermal cycler |
Drawbacks of the Sanger sequencing method
| Read Length Limitation | Sanger sequencing is limited in generating long reads compared to some modern sequencing technologies. Read lengths are typically limited to around 500-800 nucleotides. |
| Low Throughput | Sanger sequencing is a relatively low-throughput method. It involves separate reactions for each DNA fragment, making it less suitable for large-scale or high-throughput sequencing projects. |
| Labor-Intensive | Sanger sequencing involves manual steps, such as gel preparation, loading, and analysis. This makes it more labor-intensive than automated, high-throughput methods like NGS. |
| Cost-Per-Base | Sanger sequencing can be more expensive per base, especially for longer sequences. The cost per base can become a limiting factor for large-scale sequencing projects. |
| Limited Multiplexing | Multiplexing, the simultaneous sequencing of multiple samples in a single run, is limited in Sanger sequencing. NGS technologies offer much higher multiplexing capabilities. |
| Homopolymeric Regions | Sanger sequencing may have challenges in accurately determining the length of homopolymeric regions (repeating nucleotides) due to the difficulty in resolving them on the sequencing gel. |
Not Suitable for Metagenomics | Sanger sequencing is less suitable for metagenomic studies, where the goal is to sequence genetic material from multiple species within a complex sample. NGS is more adept at handling such diversity. |
Slower Turnaround Time | With its manual steps and longer individual reaction times, Sanger sequencing usually has a slower turnaround time than NGS methods. |
| Limited Dynamic Range | Sanger sequencing may face challenges in accurately quantifying variations in DNA abundance over a wide dynamic range, especially in complex samples. |
Next-Generation Sequencing (NGS) Technologies
Needed: Direct sequencing method; high-throughput, accurate, and reproducible; and cost-effective; Target: Human genome sequencing for $1000.
Benefits of NGS Technologies
- Ability to sequence thousands of genes or genomic regions simultaneously
- Ability to directly sequence unknown genomic fragments or genomes
- Capability to sequence a large number of samples in a short time
- More power to detect low-frequency variants
- Cost-effective for processing a large number of samples
References
- Sanger, F et al. “DNA sequencing with chain-terminating inhibitors.” Proceedings of the National Academy of Sciences of the United States of America vol. 74,12 (1977): 5463-7. doi:10.1073/pnas.74.12.5463.
- Moorcraft, S. Y., Gonzalez, D., & Walker, B. A. (2015). Understanding next generation sequencing in oncology: A guide for oncologists. Critical Reviews in Oncology/Hematology, 96(3), 463–474. https://doi.org/10.1016/j.critrevonc.2015.06.007.
- Valencia, C. A., Pervaiz, M. A., Husami, A., Qian, Y., & Zhang, K. (2013). Sanger sequencing principles, history, and landmarks. In SpringerBriefs in genetics (pp. 3–11). https://doi.org/10.1007/978-1-4614-9032-6_1.
