How does Fragment Analysis work? – Seq It Out #3
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 labelling 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
Host: Let’s take a look at our lab book
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 thermofisher.com/ask
and subscribe to our channel to see more videos And remember, when in doubt, just seq it out.
- Next Generation Sequencing for Gene Expression Analysis – Seq It Out #11
- How Many Samples Can I Multiplex on My NGS Run- Seq It Out #13
- Multiplex: Ethics, Emerging Technologies, Human Future – Possibilities – John Wyatt – Cape Town 2010
- Multiplex: Local Leaders – Africa Case Study – Duncan Olumbe – Cape Town 2010