Sequencing reaction is performed after the DNA template is properly prepared and quantified. Cycle sequencing or linear amplification method is used to prepare Sanger sequencing reactions. This method of preparation is different from traditional PCR methods that use two primers for exponential amplification of both DNA strands.

The sequencing reaction uses a single primer to base-pair one strand of the template and linearly amplifies the other strand of the DNA template in cycles of denaturation, annealing and amplification using Sanger’s dideoxy chain-termination method. In this process, a small fraction of the four types of fluorescently labeled dideoxynucleotides (ddATPs, ddTTPs, ddCTPs, and ddGTPs) is added as terminators to compete with their corresponding dNTPs for incorporation into synthesized DNA fragments. The elongation of DNA synthesis stops once the ddNTPs are incorporated instead of their corresponding regular dNTPs. The ladder of the synthesized DNA fragments with one nucleotide difference can then be separated on capillary electrophoresis on genetic analyzers or sequencers. Base-calling is made on the instrument software based on laser detection of fluorescent signals.

The sequencing reaction step is critical for achieving the best sequencing results. The following parameters influence sequencing quality:

Right quantity and quality of DNA template and primers

The quality and the amount of DNA template and primers determine the sequencing quality. Higher than normal level or concentration of DNA templates generates strong signals at early peak readings but the signals get weak or faded at later peak readings. If the concentration of the DNA template or primer is too low, sequencing signals may be too weak for accurate reading.

High quality of the sequencing reagents

Besides the combination of primers and templates, which differ from reaction to reaction, the rest of the sequencing components or sequencing reagents play an important role in achieving the best results. High-quality sequencing reagents, such as ADS® SupreDye™ Cycle Sequencing Kits, generate strong and uniform sequencing signals, allowing accurate sequence reading of >800 or even >900 base pairs.

The ADS™ SupreDye™ Cycle Sequencing Kits are a powerful and demonstrated alternative to BigDye™ Terminator v1.1 or v3.1 Cycling Sequencing Kits. They include kits for regular templates and high-GC templates. These kits are formulated to deliver increased robustness, even peak heights, and long read lengths.