Do you ever find yourself asking – “How does fragment analysis work and why would one perform fragment analysis in their lab?”
Let’s check it out.
Fragment analysis refers to a genetic analysis technique used for a wide variety of applications such as mutation detection, genotyping, DNA profiling, genetic mapping and linkage analysis. Various diseases, conditions and chromosomal abnormalities are detected by this method. Traditionally, the DNA fragments are separated by size in a separation matrix like agarose or polyacrylamide gels. Fragment sizes can be determined by comparing to a size standard. Then, fragments are visualized and detected either by labeling them during or after the slab gel electrophoresis using ethidium bromide dye or radioisotopes. But I think you would probably prefer doing something safer like automated capillary electrophoresis which uses fluorescent dyes and separates with higher resolution and higher accuracy.
To run fragment analysis on a capillary electrophoresis system, you can design probes and primers to flank your region of interest. Typically fluorescent dyes are attached to the primers or probes and the fragments are amplified by PCR before the electrophoresis. The ladder is usually labelled with a color that is different than the colors of the fragments. The labelled PCR products and the size marker are then electrokinetically injected into the capillaries. During electrophoresis, the negatively charged DNA fragments moves from the cathode, through the polymer-filled capillary towards the positively charged anode when high voltage is applied between the electrodes
The really cool thing about fragment analysis is that you can multiplex, meaning you can have multiple fragments in a reaction well going through the same capillary. The smaller fragments usually run faster and the bigger ones run slower. Shortly before reaching the positive electrode, the fluorescently labelled DNA fragments, separated by size, move through the path of a laser beam. The laser beam causes the dyes on the fragments to fluoresce at different emission wavelengths. A CCD camera detects the fluorescence, and the fluorescence intensities are digitalized, color-coded and displayed as peaks in the electrophoregram.
Now, that seems pretty neat right? But what’s even more amazing are the various applications for which you can use fragment analysis. One great example is the Single Nucleotide Polymorphism or SNP Genotyping. The SNaPshot Multiplex kit can investigate up to ten SNP markers simultaneously by using primers of different lengths. The primers are designed to anneal to the sequences adjacent to the ten different SNPs. Once the primer anneals, the single-base extension occurs by the addition of complementary dye terminator, or ddNTP, to the annealed primer. Each of the four ddNTPs is fluorescently labelled with a different color dye. The result is marker fragments for the different SNP alleles that are all the same length, but that vary by color.
I hope this video was helpful on fragment analysis, and I am sure you’ll have more questions.
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