They enable the use of commercially available dye-labeled sequencing primers. Also, because universal sequencing primers are efficient sequencing primers, this technique is useful with dye terminator chemistries. The disadvantage of this approach is that it adds to the cost of the PCR primers. Preparing PCR Products for Sequencing After PCR amplification, the resulting PCR product exists in the presence of PCR primers, dNTPs, enzyme and buffer components. The method used to prepare the PCR product for sequencing depends on the amounts of these components that are carried over, and on the sequencing chemistry to be used.
Different chemistries have different purification requirements.
Sequencing Dye Primer Cycle Sequencing chemistry is more forgiving with regard to PCR product purification than dye terminator chemistry. Because the sequencing primer is labeled, only fragments that are extended from the sequencing primer are detected in the gel. However, primers carried over from the amplification competes for binding and extension. If carried over in high quantities, they may cause problems. In addition to PCR primers, excess dNTPs from the amplification reaction will affect the balance of the sequencing reaction frequently resulting in decreased termination in shorter extension fragments.