SSCP analysis can be performed in an automated electrophoresis system, typically an automated DNA sequencer, which monitors the mobility of the fluorescently labeled DNA fragments during electrophoresis. PCR products can be fluorescently labeled in a number of ways. Target DNA regions can be amplified by forward and reverse primers labeled with the same single fluorescent dye or two dyes of different colors.
Post-PCR labeling can also be done easily and is more cost-effective because unlabeled primers are used. Internal labeling of PCR products is another option. The automated DNA sequencer can be gel-based or, more commonly now, capillary-based . Throughput can be increased by analysis in the same lane or capillary of multiple PCR fragments labeled with multiple colors. Mobility of DNA fragments can be standardized by inclusion in the test sample of an internal mobility standard that contains DNA fragments of known size and labeled with a color different from those of the fragments being analyzed.
Electrophoresis can be performed at ambient temperature or above because most automated DNA sequencers are equipped with a built-in heating device, but not a cooling unit. One exciting development is microchip electrophoresis, i.e., performance of electrophoresis in microchannels. Microchip SSCP analysis can be finished within a few minutes, and thus greatly reduces the analysis times by more than 100-fold when compared with conventional methods. This may revolutionize molecular genetic testing in diagnostic laboratories in the future.
No comments:
Post a Comment