The workflow of DNA fragment analysis is described in the steps below.
Primer design: For fragment analysis, two primers are utilized. One attaches to the beginning (5 end) of the target gene sequence, while the other binds to the end (3 end). To visualize the fragments, one of these primers is tagged with a fluorescent molecule (fluorophore) at its 5 end. When multiplexing, it's crucial to avoid using primers with the same fluorophore that produce fragments of similar lengths.
DNA extraction: The most suitable extraction method varies based on factors like the source or type of tissue, its handling and storage conditions prior to extraction and the manner in which the sample was collected. Common sample sources include tissue, plant substances, cells, or blood or plasma. DNA extraction can be accomplished using either commercial kits or laboratory-developed procedures designed to specific needs.
PCR amplification: Primers replicate the extracted DNA through PCR. Various PCR master mixes are available, containing a highly efficient DNA polymerase, salts like magnesium chloride (MgCl2), nucleotides and buffers. These master mixes simplify the setup of PCR reactions; other essential components include primers, template DNA, and nuclease-free water. After setting up the reactions, they are run on a thermal cycler. A typical PCR protocol starts with initial denaturation, followed by cycles of denaturation, annealing, and extension steps, and concludes with a final hold at 4°C. The temperature for annealing is crucial and should be optimized experimentally for each primer pair.
Capillary electrophoresis: Before running samples on a genetic analyzer, a plate is prepared with formamide and a series of fluorescently labeled DNA fragments of known lengths.It's crucial that this size standard covers the expected range of fragments in the experiment, ideally containing two fragments both above and below the size of each target fragment. Each sample is loaded into a separate glass capillary as part of an array. Through electrokinetic injection, the fragments are drawn into the capillaries by a high-voltage charge applied to the buffered sequencing reaction.As they migrate through the capillaries, the DNA fragments are separated based on size, with larger fragments moving more slowly through the matrix.
Data analysis: While the fragments travel through the capillary, a laser stimulates the fluorescent dye labels, causing them to emit light. These emitted signals are then visualized and represented as peaks correlating to alleles. The sizes of the fragments can also be determined during data analysis.