Low Throughput Direct Cycle Sequencing of Polymerase Chain Reaction (PCR) Products

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Traditional Polymerase Chain Reaction (PCR) product characterization relies on cloning and cycle sequencing.
• Direct sequencing of PCR products is desirable for rapid data acquisition, bypassing the cloning step.

Purpose of the Study:

• To describe a direct sequencing protocol for Polymerase Chain Reaction (PCR) products.
• To adapt gold-standard Big Dye chemistry for low-throughput sequencing on the ABI Seqstudio platform.

Main Methods:

• PCR product purification using spin column-based kits.
• Quality and quantity assessment via spectrophotometry and gel electrophoresis.
• Cycle sequencing reaction setup, amplification, and capillary electrophoresis.
• Sequence data analysis.

Main Results:

• A functional protocol for direct sequencing of PCR products was established.
• The protocol integrates established methods with modern sequencing technology for efficiency.

Conclusions:

• Direct sequencing of PCR products is a viable and efficient alternative to traditional cloning methods.
• The described protocol provides a rapid method for obtaining sequencing data from PCR amplicons using Big Dye chemistry and the ABI Seqstudio.