Since the origin of sequencing in the 1970s (see the article What is DNA sequencing?, some techniques have become extinct and a few have evolved to become the methods of choice for many researchers seeking to understand genetic variation today .
Researchers choose Sanger sequencing when performing low-throughput, targeted, or short-read sequencing. Due to its sensitivity and relative simplicity in terms of both workflow and technique, Sanger sequencing remains the gold standard in sequencing technology today and is used in a variety of applications from targeted seqencing to confirming variants identified using orthogonal methods.
Sanger sequencing utilizes a chain-termination method to provide the identity and order of nucleotide bases in a given strand of DNA. This method makes use of chemical analogues of the four nucleotide bases. These analogues, called ddNTPs, are missing the hydroxyl group that is required for extension of the polynucleotide chains that form the DNA molecule. By mixing radiolabeled ddNTPs with template DNA, strands of each possible length are produced when the ddNTPs get randomly incorporated, terminating the chain.
By the mid-1990s, researchers were performing Sanger sequencing using capillary electrophoresis. With this technology, the labeled DNA fragments are separated by size in long, thin, acrylic-fiber capillaries filled with a gel matrix. A sample containing the labeled fragments is injected electrokinetically into the capillary, and an electric field is applied to draw the fragments upward. As they pass a detection laser inside the instrument, the labels are detected, and the sequence is determined.
To learn more about Sanger sequencing, read the article What is Sanger sequencing?
Capillary electrophoresis (CE) instruments are capable of performing both Sanger sequencing and fragment analysis. Fragment analysis is a method in which DNA fragments are fluorescently labeled, separated by CE, and sized by comparison to an internal standard. While DNA sequencing by CE is used to determine the specific base sequence of a particular fragment or gene segment, this technique can also provide sizing, relative quantitation, and genotyping information for fluorescently labeled DNA fragments produced by PCR using primers designed for a specific DNA target.
Analysis of DNA fragments enables applications from cell line authentication to detection of aneuploidy. While sequencing techniques also enable these applications, researchers choose fragment analysis because it has a faster turnaround time, higher sensitivity and resolution, and is more cost-effective.
To find out more about fragment analysis, read the article What is fragment analysis?
While a number of improvements have been made to Sanger sequencing over the years, new high-throughput techniques have also arisen, termed next-generation sequencing (NGS) technologies. NGS is conducted in a massively parallel fashion.
To find out more about NGS, see the NGS Basics articles.
The spectrum of analysis of NGS can extend from a small number of genes to an entire genome, depending on the goal. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) provide the sequence of DNA bases across the genome and exome, respectively. Whole-transcriptome sequencing provides sequence information about coding and multiple noncoding forms of RNA to assess variations and gene expression levels across the entire transcriptome. Targeted sequencing covers a relatively small set of genes or targeted regions of interest. The fast turnaround time, low cost, low sample input requirement, and relative ease of interpretation make targeted sequencing particularly well suited for both translational and clinical research applications. Sanger sequencing is often used to confirm variants identified by NGS.
Figure 1. Depiction of whole-genome, whole-exome, and targeted sequencing.
Sanger sequencing, fragment analysis, and NGS enable a multitude of cutting-edge applications that are helping advance scientific understanding of genomes. To find out which technology is right for your application, see the article How do I choose the right sequencing technology?
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