To find out more about fragment analysis, read the article What is fragment analysis? 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. 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.Īnalysis of DNA fragments enables applications from cell line authentication to detection of aneuploidy. Fragment analysis is a method in which DNA fragments are fluorescently labeled, separated by CE, and sized by comparison to an internal standard. To learn more about Sanger sequencing, read the article What is Sanger sequencing?Ĭapillary electrophoresis and fragment analysisĬapillary electrophoresis (CE) instruments are capable of performing both Sanger sequencing and fragment analysis. As they pass a detection laser inside the instrument, the labels are detected, and the sequence is determined. A sample containing the labeled fragments is injected electrokinetically into the capillary, and an electric field is applied to draw the fragments upward. With this technology, the labeled DNA fragments are separated by size in long, thin, acrylic-fiber capillaries filled with a gel matrix. By mixing radiolabeled ddNTPs with template DNA, strands of each possible length are produced when the ddNTPs get randomly incorporated, terminating the chain.īy the mid-1990s, researchers were performing Sanger sequencing using capillary electrophoresis. These analogues, called ddNTPs, are missing the hydroxyl group that is required for extension of the polynucleotide chains that form the DNA molecule. This method makes use of chemical analogues of the four nucleotide bases. Sanger sequencing utilizes a chain-termination method to provide the identity and order of nucleotide bases in a given strand of DNA.

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. Researchers choose Sanger sequencing when performing low-throughput, targeted, or short-read sequencing.

Next gene sequencing