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

Learn that fragment analysis is a genetic technique used for mutation detection, genotyping, DNA profiling, genetic mapping, and linkage analysis to detect various diseases and chromosomal abnormalities.

Understand that traditionally DNA fragments were separated by size using agarose or polyacrylamide gels and visualized with ethidium bromide or radioisotopes, but automated capillary electrophoresis using fluorescent dyes is safer and more accurate.

Design probes and primers flanking the region of interest and attach fluorescent dyes to them. Amplify the fragments by PCR, ensuring the size marker ladder uses a different fluorescent color than the sample fragments.

Electrokinetically inject the labelled PCR products and size marker into the capillaries of the electrophoresis system.

Apply high voltage between electrodes to move negatively charged DNA fragments from the cathode through the polymer-filled capillary toward the positively charged anode. Smaller fragments run faster while larger fragments run slower.

Pass separated DNA fragments through a laser beam that excites the fluorescent dyes, causing them to fluoresce at different wavelengths. A CCD camera detects the fluorescence signals, which are then digitalized and color-coded.

Display the digitalized fluorescence intensities as peaks in the electrophoregram, with each peak representing a separated DNA fragment identified by size and color.

Use the SNaPshot Multiplex kit to investigate up to ten SNP markers simultaneously using primers of different lengths that anneal to sequences adjacent to SNPs. Add complementary fluorescently labeled ddNTPs to create marker fragments that are the same length but differ by color to identify different alleles.