High‐throughput gender determination using automated denaturant gel capillary electrophoresis

Abstract

Sex determination of anonymous samples is a requirement before analysis of DNA variation on X or Y chromosomes. Based on this, we designed a method for screening samples on different DNA capillary sequencing instruments with a sensitivity that is able to quantify sex chromosome abnormalities. The two different amelogenin alleles sited on the X and Y chromosomes were polymerase chain reaction amplified with the same set of primers and separated by denaturant capillary electrophoresis (DCE). Sex chromosome ratios could be reproducibly determined with a relative standard deviation of 8.7%, which is sufficient to distinguish a normal XY karyotype from an XYY karyotype associated with Klinefelter syndrome. Reconstruction experiments demonstrated sensitivity down to a simulated Y:X allelic ratio of 1:127 in all three instruments, enabling the prediction of sex chromosomal aneuploidies. When tested on anonymous pooled and single samples, DCE gave a good prediction of the male to female ratio in pools of 1000 blood donors. In conclusion, DCE is a simple and robust method for sex determination that can be readily performed on commercially available CE systems.

Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis

“…8) was major allele of 63.5 and 64.9% for PVP and LPA matrices, respectively. Given CTCE's ability to separated alleles [14][15][16][17][31][32][33] and the data presented herein the method has become a true high-throughput/low-cost method for scanning DNA for unknown variants. Based on the list price of all chemicals used to formulate a 7 or 8% PVP, one liter of polymer costs about 40$.…”

Analysis of mutational spectra by denaturing capillary electrophoresis

“…Most recently we have illustrated how the Ekstrøm's laboratory development of cycling denaturing capillary electrophoresis (CyDCE) eliminated the labor-intensive optimization steps attendant on constant denaturing systems. 26,74,76,[81][82][83][84] .…”

“…We have tested two different instruments from one of the leading manufactures of capillary DNA sequencing instruments with respect to their ability to separate alleles by DCE 76,83,89,108 . By use of standard genotyping or DNA sequencing protocols as given for the instruments manuals, except for the temperature setting, we have been able to automatically separate alleles by DCE on the single capillary instrument ABI 310 and the 16 capillary instrument ABI 3100 (figure 11).…”