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  9. Sequencing Chemistries

Sequencing Chemistries

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 (Figure 1). The products are then injected into a capillary. All current Applied Biosystems DNA sequencing kits use cycle sequencing protocols. We have focued on two general categories of sequencing chemistry approaches: Dye Primer chemistry and Dye Terminator chemistry.

Figure 1

Figure 1: Cycle Sequencing


Sequencing with Dye Primers
When using dye primer-based sequencing chemistries, four separate reactions are performed. Each reaction is labeled at its 5’ end with any one of 4 different fluorescent dyes. Each of the 4 sequencing reactions will contain either a blue, green, yellow, or red-labeled primer. The color of each reaction corresponds to either A, C, G or T. Dideoxyribonucleotides (ddNTPs) are 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 elecrophoresed on one of our capillary electrophoresis-based Genetic Analyzers (Figure 2).

Figure 2

Figure 2: One Cycle of Dye Primer Cycle Sequencing


Sequencing with Dye Terminators
Fluorescent DNA sequencing can also be performed using a chemistry in which the dyes are attached to the 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. Fluorescent fragments are generated by incorporation of dye-labeled ddNTPs. Each different ddNTP (ddATP, ddCTP, ddGTP, or ddTTP) will carry a different color of dye. All terminated fragments (those ending with a ddNTP), therefore, contain a dye at their 3’ end (Figure 3).

Figure 3

Figure 3: One Cycle of Dye Terminator Cycle Sequencing


BigDye® Cycle Sequencing Chemistries
BigDye® primers and terminators utilize 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 dye. Dichlororhodamine (dRhodamine) dyes are an improvement over conventional rhodamine dyes. dRhodamines are better spectrally resolved – there is significantly less spectral overlap at their maximum excitation wavelength, and their sequencing products show much reduced background noise. This results in a cleaner signal and greater base calling 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 suitable for most applications.

Additional Cycle Sequencing Chemistries Offered by Applied Biosystems
The dRhodamine Terminator Ready Reaction Cycle Sequencing Kit is the chemistry of choice for A-T rich samples. Alternatively, the ABI PRISM® dGTP BigDye Terminator Cycle Sequencing Kit has been optimized for GT rich, and other difficult templates.


Product Selection Guide: Sequencing Chemistry

Application BigDye® Terminators v3.1 BigDye® Terminators v1.1 dRhodamine Terminators dGTP BigDye® Terminators
G-C Rich >65% ++ ++   ++
A-T Rich >65% ++ ++ ++  
Homopolymer
A-T Rich >25 bp
    ++  
GT Rich or Difficult Templates ++ ++ + ++
de novo Sequencing
High Throughput (cDNA)
++ +    
de novo Sequencing
Low-Mid Throughput
+ ++    
Comparative Sequencing
(Germline Mutations, 50:50)
++ ++    
Comparative Sequencing
(Somatic Mutations, 10:50)
+ +    
Resequencing ++ +    
Long-Read Sequencing ++ +    
Mixed-base detection ++ +    
Sequencing short PCR products using rapid electrophoresis run modules or applications where the sequencing read begins close to the primer + ++    
BACs and Genomic DNA + +    
++ Recommended + Satisfactory