This proposal is relevant to NIMH area 115C3. The objective of our research is to improve the calibration of DNA fragment length measurement upon electrophoresis. Specifically, this technology will be applied to short tandem repeat (STR) genotyping. The primary sequence of a DNA fragment affects its electrophoretic mobility, rendering it imprecise in calibration of DNA from a different genetic locus. Complicated mechanisms have been applied to correct this error. They require multiple laser excited fluorophores, elaborate systems to differentiate their emission spectra, co-electrophoresis in external lanes of allelic ladders from loci to be typed, and increased computation time and labor. Locus specific brackets (LSB) are created through modification of alleles from each genetic locus to be measured. Closely related in both sequence and length to the alleles of their loci of origin, they provide compatible electrophoretic mobility for calibration of fragment length measurement. A quadriplex of LSB will be developed for PCR amplification and calibration of the alleles of 4 STR loci co-electrophoresed in a single lane, and directed toward use with less complicated and expensive detection systems. Accurate, less expensive, user friendly calibration will aid in the dissemination of STR typing.
The complexity and cost of the current STR analysis technology hinders its wide deployment. Only 11,000 of 425,000 samples entered onto the CODIS national DNA database of violent crimes have been STR genotyped. There are currently 750,000 pending paternity cases in the US. Recent welfare reform legislation passed by Congress may increase the need for paternity testing. LSB will allow cost effective instruments to perform adequate work, reduce the complexity and shorten the learning curve of users. Furthermore, LSB open a new window for clinical DNA genotyping.
