How does Fragment Analysis work? - Seq It Out #3

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How does fragment analysis work and why would one perform fragment analysis in their lab? Stay tuned…..
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. Submit your question at www.thermofishe... and subscribe to our channel to see more videos like this.
And remember, when in doubt, just Seq It Out

[@tonderaigumbo9846]

You guys are amazing👊

[@mohammadsohail2279]

Quite useful ! Thankyou

[@melissaespinoza827]

Sure does help. Great graphics :)

[@thermofisher]

Thank you for watching! Glad it helped.

[@ajay1rana]

Where does the fragment finally goes from cathode? In the anode buffer?

[@thermofisher]

Thank you for your question. Please reach out to our technical support team at thermofisher.com/askaquestion. Thank you!

[@taratara3986]

I have one constructive criticism: the scientist in the video speaks way too fast! Speaking at a reasonable speed is very important in effective communication.

[@martjenk]

Ok but I think it's a personal thing. I find some people speak too slowly and sometimes I "switch off". Under the settings icon in the video you can slow down (or speed) up the speed of the video.

[@taratara3986]

​@@martjenk Thanks for the advice on speed settings; I changed it from 'Normal' to '0.75' and it actually worked!

[@ketanmusic2140]

Is fragment analysis and multiplex ligation dependent probe amplification same?

[@Fire-Swan]

+Ketan Makwana MLPA isnt the as fragment analysis. MLPA is normally used to see if entire exons are deleted or inserted. Fragment analysis is normally used for when there are many short tandem repeats which would be problematic to perform a standard PCR on for various reasons such as having primer specificity.

[@eduardomorgado1488]

i hate genetics

[@eduardomorgado1488]

good video tho