What is fragment analysis?

Fragment analysis is a genetic analysis method comprising a series of techniques in which DNA fragments are fluorescently labeled, separated by capillary electrophoresis (CE), and sized by comparison to an internal standard.

CE-based genetic analyzers are capable of performing both Sanger sequencing and fragment analysis. In contrast to Sanger sequencing, fragment analysis can provide sizing, relative quantitation, and genotyping information using fluorescently labeled DNA fragments produced by PCR using primers designed for a specific DNA target. This information enables researchers to detect differences in alleles, homo- and heterozygosity, chimerism, sample mixtures, and inheritance. Fragment analysis enables a wide variety of applications, including cell line authentication, determination of CRISPR-Cas9 genome editing efficiency, microsatellite marker analysis, SNP genotyping, and more. Fragment analysis has a fast turnaround time, high sensitivity and resolution, and is cost-effective.

Additional advantages of fragment analysis include:

• Multiplexing—alleles for overlapping loci are distinguished by labeling locus-specific primers with different colored dyes; due to this ability, fragment analysis enables the analysis of more than 20 loci in a single reaction
• Sensitivity—fragments differing by only one base pair are accurately sized
• Simple preparation—no DNA cleanup is required (as opposed to sequencing)
• Easy data analysis—genetic analysis software simplifies data analysis
• Independent method—does not require knowledge of the sequence of the fragment
• Relative quantitation information—from peak intensity measurements
• Automated workflows—ability to run thousands of DNA fragment samples in one day
• High throughput—can be achieved using capillary arrays

The DNA fragment analysis workflow consists of four general steps: DNA extraction, PCR amplification, capillary electrophoresis, and data analysis (Figure 1).

DNA extraction is a critical first step in the experimental workflow of DNA fragment analysis. The overall efficiency, quality, and size of the PCR product can be significantly affected by characteristics of the sample itself and the method chosen for nucleic acid extraction and purification. Ideal methods will vary depending on the source or tissue type, how the sample was obtained from its source, and how the sample was handled or stored prior to extraction.

To perform fragment analysis on a CE system, primers must be designed that flank the region of interest. Fluorescent dyes are attached to the primers, and the fragments are amplified by PCR before electrophoresis.

To prepare for capillary electrophoresis, a spectral calibration with the corresponding matrix standard for the selected group of dyes must be performed on the genetic analyzer in order to accurately detect the dye-labeled primers. Each unknown sample is mixed with the size standard and formamide before proceeding with electrophoresis. Size standards allow sizing of sample peaks and correct for injection variations.

During capillary electrophoresis, the products of the PCR are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system.

Data analysis software provides a profile of the separation, precisely calculates the sizes of the fragments, and determines the microsatellite alleles present in the sample (Figure 2).

Learn more about the steps of the fragment analysis workflow ›

Fragment analysis enables many applications and methods, including:

• Cell line authentication—many journals and funding agencies now require researchers to ascertain that the cell lines they use are authentic; fragment analysis can provide a simple, inexpensive, and highly specific genetic “fingerprint” of a cell line
• Determination of CRISPR-Cas9 genome editing efficiency—in any genome editing experiment, the repair process is not completely efficient or accurate; fragment analysis can be used to screen primary transformed cultures to determine editing efficiency
• Single-nucleotide polymorphism (SNP) genotyping—SNPs consist of single–nucleotide base changes that result in up to four different alleles at a given locus, and have been shown to be responsible for genetic traits, susceptibility to disease, and response to drug therapies; fragment analysis can distinguish between these when dye-labeled nucleotides are used in place of a labeled primer during the PCR step
• Microsatellite marker analysis—microsatellite markers are codominant, polymorphic DNA loci containing repeated nucleotide sequences, typically with 2 to 7 nucleotides per repeat unit; the number of nucleotides in the repeated unit is the same for the majority of the repeats within an individual microsatellite locus, but the number of repeats for a specific locus may differ, resulting in alleles of varying length, which can be analyzed with fragment analysis and used to identify individuals (for example, for conservation or human identification (HID) purposes)

Fragment analysis is a powerful research tool that provides relative quantitation, sizing, and genotyping information and enables a wide array of genetic analysis applications.

To find out more about the applications enabled by CE, see the article What applications does capillary electrophoresis enable?