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.
Developed by Frederick Sanger and colleagues in 1977, it was the most widely used sequencing
method for approximately 25 years.
More recently, Sanger sequencing has been supplanted by "Next-Gen" sequencing methods,
especially for large-scale, automated genome analyses.
However, the Sanger method remains in wide use, primarily for smaller-scale projects
and for obtaining especially long contiguous DNA sequence reads.
Method The classical chain-termination method requires
a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleosidetriphosphates,
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 labeled 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 three other
nucleotides are ordinary ones.
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, is now being used for the vast majority of sequencing projects.
Automation and sample preparation
Automated DNA-sequencing instruments can sequence up to 384 DNA samples in a single batch in
up to 24 runs a day.
DNA sequencers carry out capillary electrophoresis for size separation, detection and recording
of dye fluorescence, and data output as fluorescent peak trace chromatograms.
Sequencing reactions by thermocycling, cleanup and re-suspension in a buffer solution before
loading onto the sequencer are performed separately.
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 below visual examination by a human operator, but sufficient
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 hetroduplex
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
In the TC reaction chamber, dye-terminator sequencing reagent, template DNA, and primers
are loaded into the TC chamber and thermal-cycled for 35 cycles.
Purification The charged reaction mixture is conducted
through a capture/purification chamber at 30°C via a 33-Volts/cm electric field applied
between capture outlet and inlet ports.
The capture gel through which the sample is driven, consists of 40 μM of oligonucleotide
covalently bound to a polyacrylamide matrix.
Extension fragments are immobilized by the gel matrix, and excess primer, template, free
nucleotides, and salts are eluted through the capture waste port.
The capture gel is heated to 67-75°C to release extension fragments.
Capillary electrophoresis Extension fragments are injected into the
CE chamber where they are electrophoresed through a 125-167-V/cm field.
Platforms The Apollo 100 platform integrates the first
two Sanger sequencing steps in a fully automated system.
The manufacturer claims that samples are ready for capillary electrophoresis within three
hours of the sample and reagents being loaded into the system.
The Apollo 100 platform requires sub-microliter volumes of reagents.
Comparisons to other sequencing techniques The ultimate goal of high-throughput sequencing
is to develop systems that are low-cost, and extremely efficient at obtaining extended
read lengths.
Longer read lengths of each single electrophoretic separation, substantially reduces the cost
associated with de novo DNA sequencing and the number of templates needed to sequence
DNA contigs at a given redundancy.
Microfluidics may allow for faster, cheaper and easier sequence assembly.
References
Further reading http:nano.cancer.govnanotech_news_2006-05-30a.asp
http:nano.cancer.govmonthly_feature_2005_aug.asp External links
MBI Says New Tool That Automates Sanger Sample Prep Cuts Reagent and Labor Costs