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Sanger sequencing by capillary electrophoresis is the gold-standard DNA sequencing technique that is used in a number of experimental workflows in life sciences laboratories. 

View the tabs below to get an overview of the Sanger method of DNA sequencing, to learn about cycle sequencing and fluorescent labeling chemistries used for Sanger sequencing, and to understand the capillary electrophoresis process to separate DNA fragments.

Sanger sequencing technology overview

DNA Strand Synthesis by Formation of Phosphodiester Bonds Enlarge Image
Figure 1: DNA strand synthesis by formation of phosphodiester bonds.

During Sanger sequencing, DNA polymerases copy single-stranded DNA templates by adding nucleotides to a growing chain (extension product). Chain elongation occurs at the 3' end of a primer, an oligonucleotide that anneals to the template. The deoxynucleotides added to the extension product are selected by base-pair matching to the template.

The extension product grows by the formation of a phosphodiester bridge between the 3'-hydroxyl group on the primer and the 5'-phosphate group of the incoming deoxynucleotide (Watson et al. 1987). Growth occurs in the 5' -> 3' direction (Figure 1).

DNA polymerases can also incorporate analogues of nucleotide bases. The dideoxy method of DNA sequencing developed by Sanger et al. 1977 takes advantage of this characteristic by using 2',3'-dideoxynucleotides as substrates. When dideoxynucleotides are incorporated at the 3' end of the growing chain, chain elongation is terminated selectively at A, C, G, or T. This is because once the dideoxynucleotide is incorporated, the chain lacks a 3'-hydroxyl group so further elongation of the chain is prevented.

DNA Sequencing

Learn more about DNA sequencing by watching this video.

Cycle sequencing

Cycle sequencing Enlarge Image
Figure 1: Cycle sequencing.

Cycle sequencing is a simple method in which successive rounds of denaturation, annealing, and extension in a thermal cycler result in linear amplification of DNA extension products (Figure 1). The products are then injected into a capillary for electrophoretic separation. All current Applied Biosystems® DNA sequencing kits use cycle sequencing protocols with two different chemistries: dye primer chemistry or dye terminator chemistry.

Sequencing with dye primers

Four separate reactions are performed when using dye primer-based sequencing chemistry. Each reaction contains a primer labeled at its 5' end with one of 4 different fluorescently-labeled dyes. The blue, green, yellow, or red-labeled primers corresponding to each of the 4 nucleotides–A, C, G or T. Dideoxynucleotides (ddNTPs) are also present in each reaction mix, and randomly terminate DNA synthesis, creating DNA fragments of varying lengths. Since a fluorescently-labeled primer is used for extension, all terminated fragments are fluorescently labeled. Following a sufficient number of cycles to allow for optimal generation of extended products, the four reactions are combined and separated on one of our capillary electrophoresis-based genetic analyzers (Figure 2). 

Sequencing with dye terminators

One Cycle of Dye Primer Cycle Sequencing Enlarge Image
Figure 2: One cycle of dye primer cycle sequencing.

Fluorescent DNA sequencing can also be performed by directly attaching a different dye to each of the four ddNTPs, thereby requiring only one reaction tube per sample, instead of four. Since only one reaction tube is required for the dye terminator reaction, this chemistry is simpler to use than dye-primer chemistry. DNA template, unlabeled primer, buffer, the four dNTPs, the four fluorescently-labeled ddNTPs, and AmpliTaq® DNA Polymerase are added to the reaction tube. Fluorescently-labeled fragments are generated by incorporation of the dye-labeled ddNTPs. All terminated fragments (those ending with a ddNTP), therefore, contain a dye at their 3' end (Figure 2). The fragments are then separated by capillary electrophoresis.

BigDye® cycle sequencing chemistries

One cycle of dye terminator cycle sequencing. Enlarge Image
Figure 3: One cycle of dye terminator cycle sequencing.

BigDye® primers and terminators use single energy transfer molecules which include an energy donor and acceptor dye connected by a highly efficient energy transfer linker. In the structure of the BigDye® molecule, the acceptor is a dichlororhodamine (dRhodamine) dye, which offers advantages over conventional rhodamine dyes. dRhodamines have better spectral resolution, with significantly less spectral overlap at their maximum excitation wavelength, producing sequencing products with greatly reduced background noise. This modification results in a cleaner signal and greater basecalling accuracy at longer read lengths. An energy transfer linker couples the donor fluorescein and acceptor dRhodamine dyes for efficient energy transfer in a single dye molecule. These brighter, cleaner dyes result in a sequencing chemistry that is suitable for most applications. 

Additional Applied Biosystems® cycle sequencing chemistries

The dRhodamine Terminator [RK1] Cycle Sequencing Ready Reaction Kit is the chemistry of choice for AT-rich samples. Alternatively, the dGTP BigDye® Terminator v3.0 Ready Reaction Cycle Sequencing Kit has been optimized for GT-rich, and other difficult templates.

Fluorescently labeled DNA fragments move through a capillary. Enlarge Image
Figure 1: Fluorescently labeled DNA fragments move through a capillary.

During capillary electrophoresis, the products of the cycle sequencing reaction are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the negatively-charged DNA fragments move through the polymer in the capillaries toward the positive electrode (Figure 1).Capillary electrophoresis can resolve DNA molecules that differ in molecular weight by only one nucleotide.

Shortly before reaching the positive electrode, the fluorescently-labeled DNA fragments, separated by size, move through the path of a laser beam. The laser beam causes the dyes on the fragments to fluoresce. An optical detection device on Applied Biosystems® genetic analyzers detects the fluorescence signal (Figure 2).

DNA fragmentsEnlarge Image
Figure 2: DNA fragments pass through a laser beam and optical detector.

The data collection software converts the fluorescence signal to digital data, and records the data in a *.ab1 file. Because each dye emits light at a different wavelength when excited by the laser, all four colors, and therefore, all four bases, can be detected and distinguished in one capillary injection.

What is cycle sequencing?

Linear amplification of extension products Enlarge Image
Figure 1: Linear amplification of extension products.

Cycle sequencing is a simple method in which successive rounds of denaturation, annealing, and extension in a thermal cycler result in linear amplification of extension products (see Figure 1). The products are then loaded onto a gel or injected into a capillary. All current BigDye sequencing kits use cycle sequencing protocols.

What are the advantages of cycle sequencing?

The advantages are as follows:

  • Protocols are robust and easy to perform
  • Cycle sequencing requires much less template DNA than single-temperature extension methods
  • Cycle sequencing is more convenient than traditional single-temperature labeling methods that require a chemical denaturation step for double-stranded templates
  • High temperatures reduce secondary structure, allowing for more complete extension
  • High temperatures reduce secondary primer-to-template annealing
  • The same protocol is used for double- and single-stranded DNA
  • The protocols work well for direct sequencing of PCR products
  • Difficult templates, such as bacterial artificial chromosomes (BACs), can be sequenced

What is dye terminator cycle sequencing?

Features of dye-labeled terminator reactions. Enlarge Image
Figure 2: Features of dye-labeled terminator reactions.

With dye terminator labeling, each of the four dideoxy terminators (ddNTPs) is tagged with a different fluorescent dye. The growing chain is simultaneously terminated and labeled with the dye that corresponds to that base (see Figure 2). An unlabeled primer can be used. Dye terminator reactions are performed in a single tube. They require fewer pipetting steps than dye primer reactions. Four-color dye labeled reactions are loaded in a single gel lane or capillary injection. False stops, i.e., fragments that are not terminated by a dideoxynucleotide, go undetected because no dye is attached.

What is dye primer cycle sequencing?

Features of dye-labeled primer reactions Enlarge Image
Figure 3: Features of dye-labeled primer reactions.

With dye primer labeling, primers are tagged with four different fluorescent dyes. Labeled products are generated in four separate base-specific reactions. The products from these four reactions are then combined and loaded into a single gel lane or capillary injection (see Figure 3). Dye primer chemistries generally produce more even signal intensities than dye terminator chemistries.

Labeled primers are available for common priming sites. Custom primers can also be labeled. Four-color dye-labeled reactions are loaded onto a single lane or capillary injection.

What are matrix standards?

The precise spectral overlap between the four dyes is measured by running DNA fragments labeled with each of the dyes in a special calibration run on the Applied Biosystems Genetic Analyzer. These dye-labeled DNA fragments are called matrix standards.

The Data Utility software then analyzes the data from the matrix standard samples and creates a matrix file. These numbers are normalized fluorescence intensities and represent a mathematical description of the spectral overlap that is observed between the dyes.

The matrix files in an instrument file are used for specific types of chemistry, and provide information to the Sequencing Analysis software to allow it to correct for spectral overlap.

What is De Novo sequencing?

The initial generation of the primary genetic sequence of a particular organism is called de novo sequencing. A detailed genetic analysis of any organism is possible only after de novo sequencing has been performed. de novo sequencing is typically accomplished by assembling individual sequence reads into longer contiguous sequences (contigs) or correctly ordered contigs (scaffolds) in the absence of a reference sequence.
More information on de novo sequencing ›

What is resequencing?

Resequencing specific genomic regions is commonly performed to indentify the mutations and changes in genes. Resequencing techniques can be focused on known mutations (genotyping) or used to search for any mutation in the target DNA region (variant analysis).
More information on targeted resequencing ›

What is SNP analysis?

Single nucleotide polymorphism (SNP) is the substitution of one base for another. They are common DNA variants present across the human genome and have been shown to be responsible for differences in genetic traits, susceptibility to disease, and response to drug therapies. Genotyping of SNPs has become extremely important to researchers working to understand and treat disease. SNPs occur approximately once every 100 to 300 bases and can be detected by various different techniques such as sequencing, SNapShot, and more.
More information on SNP analysis ›

What is heterozygote detection?

diploid organism is heterozygous at a gene locus when its cells contain two different alleles of a gene. Heterozygotes are essentially detected by sequencing (SNP and small deletion-insertion) or gene copy number (big deletion-insertion).
More information on heterozygote detection ›

What is BAC end-sequencing?

Bacterial artificial clones (BACs) are large segments (100kb-200kb) of DNA cloned into bacteria from another species. Multiple copies can be made after cloning. Sequences from the BAC ends provide highly specific markers. These sequences can then be queried against BAC libraries for confirmation.

What does "Checking Clone Constructs" mean?

This refers to verifying that the DNA of interest has been properly cloned into the vector by sequencing.

What is multicomponent analysis?

Multicomponent analysis Enlarge Image
Figure 4: Multicomponent analysis.

Multicomponent analysis is the process that separates the four different fluorescent dye colors into distinct spectral components. Although each of these dyes emits its maximum fluorescence at a different wavelength, there is some overlap in the emission spectra between the four dyes (see Figure 4). The goal of multicomponent analysis is to isolate the signal from each dye so that there is as little noise in the data as possible.

What is HLA typing?

The human leukocyte antigen test (HLA) detects antigens (genetic markers) in white blood cells. The 4 types of human leukocyte antigens are: HLAA, HLAB, HLAC, and HLAD. The HLA test checks the tissue compatability and recipient / donor tissue typing. It is also used in genetic counseling and paternity testing. (Research only)
More information on HLA typing by sequencing ›

What is methylation detection?

DNA methylation occurs at CpG sites, which are DNA sequences in which cytosine lies next to guanine. Methylation is mediated by an enzyme (DNA methyltransferase). CpG sites are rare in an eukaryotic genome, except in regions near the promoter of a gene. These regions are known as CpG islands, and the state of methylation at these CpG sites is critical for gene activity/expression.
More information on methylation analysis ›

What is mtDNA sequencing?

The common abbreviation for mitochondria is mtDNA. Mitochondiral molecules are present in 100s-1000s of copies per cell, as opposed to the nuclear DNA, which is present in just two copies per cell. The abundance of mtDNA allows discrimination among individuals or biological samples, particularly if nuclear DNA is degraded or unavailable.
More information on mitochondrial sequencing ›

What is comparative genomic resequencing?

Comparative Genomic Resequencing is the comparison of genomes and individuals within a genome. Comparative genomics makes possible the application of information gained from a sample genome to a more complex genome. It is the basis for the understanding of genetic variation in a population.

What is the SAGE™ method?

SAGE™ is a method for quantitative, genome-wide gene expression pattern analysis. A short sequence tag (10-25 bp) contains sufficient information to identify a transcript.