SANGER SEQUENCING

SANGER SEQUENCING

Overview – What is Sanger sequencing?

Sanger sequencing, also known as chain-termination sequencing or dideoxy sequencing has been the powerhouse of DNA sequencing since its invention in the 1970s. The process is based on the detection of labelled chain-terminating nucleotides that are incorporated by a DNA polymerase during the replication of a template. 

Schematic overview Sanger sequencing

The method has been extensively used to advance the field of functional and comparative genomics, evolutionary genetics and complex disease research. Notably, the dideoxy method was employed in sequencing the first human genome in 2002. Because of its suitability for routine validation of cloning experiments and PCR fragments, Sanger sequencing remains a popular technique in many laboratories across the world. 

Applications – What are the advantages of Sanger sequencing?

Sanger DNA sequencing is widely used for research purposes like:

  • Targeting smaller genomic regions in a larger number of samples
  • Sequencing of variable regions
  • Validating results from next-generation sequencing (NGS) studies
  • Verifying plasmid sequences, inserts, mutations 
  • HLA typing
  • Genotyping of microsatellite markers
  • Identifying single disease-causing genetic variants

Workflow – Sanger sequencing methods & technologies

Dideoxy sequencing is based on synthesis of DNA strands that are complementary to a template DNA strand. The sequencing reaction uses normal deoxynucleoside triphosphates (dNTPs) and modified dideoxynucleoside triphosphates (ddNTPs) for strand elongation. The ddNTPs are chemically altered with a fluorescent label and with a chemical group that inhibits phosphodiester bond formation, causing DNA polymerase to stop DNA extension whenever a ddNTP is incorporated. The resulting DNA fragments are subjected to capillary electrophoresis, where the fragments flow through a gel-like matrix at different speeds according to their size. Each of the four modified ddNTPs carries a distinct fluorescent label. The emitted fluorescence signal from each excited fluorescent dye determines the identity of the nucleotide in the original DNA template. 

Sanger sequencing versus next-generation sequencing

The DNA sequencing field did not stop evolving with the successful adaptation of Sanger sequencing. The establishment of next-generation sequencing (NGS) and third-generation sequencing technologies offered substantial benefits compared to the traditional dideoxy method. However, the chain-termination method remains widely used in the sequencing field, because it offers several distinct advantages. Specifically, Sanger sequencing is preferable over NGS for:

  • Sequencing of single genes
  • Cost-efficient sequencing of single samples 
  • Verification sequencing for site-directed mutagenesis or the presence of cloned inserts
  • In some cases, analysis of longer fragments (~1,000 bp in length)
  • In some cases, less error-prone than NGS

Nevertheless, next-generation sequencing is often considered to be superior to Sanger sequencing, especially for project objectives that require:

  • Cost-efficient, simultaneous interrogation of more than 100 genes at a time
  • Finding novel variants by increasing the number of targets sequenced per run
  • Analysis of samples with low-input DNA
  • Sequencing of whole genomes, especially microbial genomes

Often laboratories rely on both Sanger and NGS techniques, where Sanger sequencing is used for more routine small-scale projects and NGS is applied to meet large-scale sequencing needs.  

Scientific expertise: Sanger sequencing

GATC Biotech has its roots deep in the Sanger sequencing field. Founded in 1990, the company commercialised the first nonradioactive sequencing technology platform, GATC1500, which was used for the sequencing of the first Saccharomyces cerevisiae genome.

In 1996, the company began offering sequencing services, establishing several industry milestones along the way. 

To this day, the company maintains a leading position as a Sanger sequencing provider. In the past few years, the company’s Sanger services have helped elucidate a wide range of research questions on topics ranging from membrane trafficking to CRISPR interference to tumour mutations to the composition of microbial communities and much more. The company also specialises in custom applications of Sanger sequencing, including:

  • PCR establishment with or without primer design
  • Cloning
  • DNA isolation
  • Bioinformatics: SNP/InDel analysis, assembly, mapping
  • Sanger sequencing according to DIN EN ISO/IEC 17025
  • Genotyping – Determining the genetic variants (SNPs/InDels) of an individual for identity testing, pharmacogenomics, disease screening, population genetics and DNA sample profiling purposes; can include Restriction Fragment Length Polymorphism (RFLP) analysis
  • Primer walking – for sequencing long DNA templates (up to 7 kb) from end to end by a process that includes several rounds of sequencing, with each round initiated from a new primer based on the previously determined sequence; applications include gap filling, sequencing of large clones and sequence verification

Publications

Find here, a list of selected research articles supported by GATC Biotech’s sequencing products, as well as the latest publications on Sanger sequencing.

Products related to Sanger sequencing

GATC Biotech’s Sanger sequencing products can process a variety of starting materials submitted in tubes or plates. Exceptionally high-quality sequencing results are available online within hours via myGATC. 

Rely on our established LIGHTRUN product for DNA samples premixed with primer and enjoy sequencing reads up to 1,100 nt in length in Phred20 quality. Available with the NightXpress option for the fastest turnaround times possible.

For the most difficult templates, take advantage of our SUPREMERUN service with optimised protocols for GC-rich sequences, hairpins and secondary structure features that often inhibit sequencing reactions. Request SNP and InDel analysis for free. Available with NightXpress option to maximise the speed of data delivery.

Additional services for customised Sanger projects like cloning, DNA isolation, primer design, primer walking and genotyping, including fragment length analysis, are available upon request. 

Further reading on Sanger sequencing

Heller, C. Principles of DNA separation with capillary electrophoresis. Electrophoresis. 22(4), 629 – 643 (2001).

Sanger, F., Nicklen, S., Coulson, A.R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 74(12), 5463 - 7 (1977).