DNA Sequencing by Capillary Electrophoresis

Introduction

For over 25 years, Applied Biosystems has been a pioneer in the field of genetic analysis by offering systems to address the expansion of genetic analysis applications and the evolving needs of today’s research environment.

Supporting life science researchers in Academia and Industry, addressing a wide range of applications and throughput needs, Applied Biosystems is proud to provide gold-standard instrumentation, reagents, consumables, analysis software, and world-class technical support to address a limitless progression of scientific questions, leading to answers and solutions.

DNA Sequencing by Capillary Electrophoresis is a key technology in a number experimental workflows in the life science laboratory. If you are new to the techniques involved in DNA Sequencing, click the topics in the left sidebar of the page.

DNA Extraction

DNA extraction is a critical first step in the experimental workflow of DNA sequence analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction.

Isolating high-quality DNA from various sample types can be challenging, and ideal methods will vary depending on the tissue type (including blood), how it was obtained from its source, and how the sample was handled or stored prior to extraction. Methods for nucleic acid isolation are often accomplished by mechanical disruption or chemical methods, which are sometimes automated.

Whether manual or automated methods of extraction are used, care should be taken to minimize subsequent or further degradation of the DNA, by avoiding exposure to heat, light, freeze-thaw cycles, and vortexing. Furthermore, when extracting DNA from multiple samples, the laboratory setup should minimize the propensity for possible cross-contamination between samples.

Primer Design & Amplification

Many common DNA sequencing workflows require you to amplify your extracted DNA sample before sequencing. To amplify your DNA sample, you need DNA polymerase (such as AmpliTaq® Gold DNA Polymerase), nucleotides (dNTPs), reaction buffer, primers, and a thermal cycler.

We offer several tools to help streamline this important step. Our VariantSEQr™ Resequencing System enables you to resequence human disease genes and other targets of common interest. Our mitoSEQr™ Resequencing System enables you to discover variants in the human mitochondrial genome. Both systems provide:

Ready-to-use resequencing sets

　　Suggested protocols for PCR and sequencing

　　A project template for SeqScape® Software

　　A gene information file/data sheet

Methyl Primer Express™ Software v1.0 is a free software tool that enables you to design high-quality PCR primers for methylation mapping experiments. Simply cut and paste in your region of interest. The tool searches for CpG islands and simulates bisulfite modification of DNA in silico.

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

Sample Cleanup

After the sequencing reaction, it is important to remove unincorporated dye terminators and salts that may compete for capillary electrophoretic injection. Unincorporated terminators can co-migrate with the sequencing template, resulting in basecalling errors, and excess salt translates to poor signal-to-noise ratios.

Electrophoresis

After performing the post-sequencing reaction purification, samples are ready for analysis on an Applied Biosystems capillary electrophoresis-based genetic analyzer.

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.

Data Analysis

After electrophoresis, Data Collection software creates a Sample File of the raw data. Using downstream software applications, further data analysis is required to translate the collected color-data images into the corresponding nucleotide bases.

Frequently Asked Questions

What is Sanger Dideoxy Sequencing and how is it different from Applied Biosystems Fluorescent Sequencing? What is Dye Terminator Cycle Sequencing? What are Matrix Standards? What is BAC-End Sequencing?  For the answers to these questions and more, read our DNA Sequencing FAQs.