Sanger sequencing
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederick Sanger and colleagues in 1977, it became the most widely used sequencing method for approximately 40 years. It was first commercialized by Applied Biosystems in 1986. More recently, higher volume Sanger sequencing has been replaced by "Next-Gen" sequencing methods, especially for large-scale, automated genome analyses. However, the Sanger method remains in wide use, for smaller-scale projects, and for validation of Next-Gen results. It still has the advantage over short-read sequencing technologies in that it can produce DNA sequence reads of > 500 nucleotides.
Method
The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates, and modified di-deoxynucleotidetriphosphates, the latter of which terminate DNA strand elongation. These chain-terminating nucleotides lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently labelled for detection in automated sequencing machines.The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides, while the other added nucleotides are ordinary ones. The dideoxynucleotide concentration should be approximately 100-fold higher than that of the corresponding deoxynucleotide to allow enough fragments to be produced while still transcribing the complete sequence. Putting it in a more sensible order, four separate reactions are needed in this process to test all four ddNTPs. Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. In the original publication of 1977, the formation of base-paired loops of ssDNA was a cause of serious difficulty in resolving bands at some locations. This is frequently performed using a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes. The DNA bands may then be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide. The relative positions of the different bands among the four lanes, from bottom to top, are then used to read the DNA sequence.
Technical variations of chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled at the 5' end with a fluorescent dye. Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation. The later development by Leroy Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.
Chain-termination methods have greatly simplified DNA sequencing. For example, chain-termination-based kits are commercially available that contain the reagents needed for sequencing, pre-aliquoted and ready to use. Limitations include non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence, and DNA secondary structures affecting the fidelity of the sequence.
Dye-terminator sequencing
Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths.Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis.
This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, was used for the vast majority of sequencing projects until the introduction of Next Generation Sequencing.
Automation and sample preparation
Automated DNA-sequencing instruments can sequence up to 384 DNA samples in a single batch. Batch runs may occur up to 24 times a day. DNA sequencers separate strands by size using capillary electrophoresis, they detect and record dye fluorescence, and output data as fluorescent peak trace chromatograms. Sequencing reactions, cleanup and re-suspension of samples in a buffer solution are performed separately, before loading samples onto the sequencer. A number of commercial and non-commercial software packages can trim low-quality DNA traces automatically. These programs score the quality of each peak and remove low-quality base peaks. The accuracy of such algorithms is inferior to visual examination by a human operator, but is adequate for automated processing of large sequence data sets.Challenges
Common challenges of DNA sequencing with the Sanger method include poor quality in the first 15-40 bases of the sequence due to primer binding and deteriorating quality of sequencing traces after 700-900 bases. Base calling software such as Phred typically provides an estimate of quality to aid in trimming of low-quality regions of sequences.In cases where DNA fragments are cloned before sequencing, the resulting sequence may contain parts of the cloning vector. In contrast, PCR-based cloning and next-generation sequencing technologies based on pyrosequencing often avoid using cloning vectors. Recently, one-step Sanger sequencing methods such as Ampliseq and SeqSharp have been developed that allow rapid sequencing of target genes without cloning or prior amplification.
Current methods can directly sequence only relatively short DNA fragments in a single reaction. The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide.
Microfluidic Sanger sequencing
Microfluidic Sanger sequencing is a lab-on-a-chip application for DNA sequencing, in which the Sanger sequencing steps are integrated on a wafer-scale chip using nanoliter-scale sample volumes. This technology generates long and accurate sequence reads, while obviating many of the significant shortcomings of the conventional Sanger method by integrating and automating the Sanger sequencing steps.In its modern inception, high-throughput genome sequencing involves fragmenting the genome into small single-stranded pieces, followed by amplification of the fragments by Polymerase Chain Reaction. Adopting the Sanger method, each DNA fragment is irreversibly terminated with the incorporation of a fluorescently labeled dideoxy chain-terminating nucleotide, thereby producing a DNA “ladder” of fragments that each differ in length by one base and bear a base-specific fluorescent label at the terminal base. Amplified base ladders are then separated by Capillary Array Electrophoresis with automated, in situ “finish-line” detection of the fluorescently labeled ssDNA fragments, which provides an ordered sequence of the fragments. These sequence reads are then computer assembled into overlapping or contiguous sequences which resemble the full genomic sequence once fully assembled.
Sanger methods achieve read lengths of approximately 800bp. The longer read lengths in Sanger methods display significant advantages over other sequencing methods especially in terms of sequencing repetitive regions of the genome. A challenge of short-read sequence data is particularly an issue in sequencing new genomes and in sequencing highly rearranged genome segments, typically those seen of cancer genomes or in regions of chromosomes that exhibit structural variation.
Applications of microfluidic sequencing technologies
Other useful applications of DNA sequencing include single nucleotide polymorphism detection, single-strand conformation polymorphism heteroduplex analysis, and short tandem repeat analysis. Resolving DNA fragments according to differences in size and/or conformation is the most critical step in studying these features of the genome.Device design
The sequencing chip has a four-layer construction, consisting of three 100-mm-diameter glass wafers and a polydimethylsiloxane membrane. Reaction chambers and capillary electrophoresis channels are etched between the top two glass wafers, which are thermally bonded. Three-dimensional channel interconnections and microvalves are formed by the PDMS and bottom manifold glass wafer.The device consists of three functional units, each corresponding to the Sanger sequencing steps. The Thermal Cycling unit is a 250-nanoliter reaction chamber with integrated resistive temperature detector, microvalves, and a surface heater. Movement of reagent between the top all-glass layer and the lower glass-PDMS layer occurs through 500-μm-diameter via-holes. After thermal-cycling, the reaction mixture undergoes purification in the capture/purification chamber, and then is injected into the capillary electrophoresis chamber. The CE unit consists of a 30-cm capillary which is folded into a compact switchback pattern via 65-μm-wide turns.
Sequencing chemistry
;Thermal cycling;Purification
;Capillary electrophoresis