Anomalous Separation of Small Y-Chromosomal DNA Fragments on Microchip Electrophoresis

We investigated an anomalous DNA separation where two DNA fragments from the human Y-chromosome sY638 (64 bp) and sY592 (65 bp), with only one base pair difference, were separated. This result is abnormal since in a previous study, we found that 5 bp was the minimum difference between two DNA fragments that the microchip electrophoresis system can separate. The formation of a mini-loop in the structure of the DNA fragment of sY638 (64 bp) was strongly expected to be the reason. To investigate this, we synthesized three modified DNA fragments for sY638 (64 bp), and the modifications were in two expected locations for possible mini-loop formation. Later, the separation between sY592 (65 bp) and the three modified fragments of sY638 (64 bp) was not possible. Thus, we conclude that the formation of a mini-loop in the structure of the DNA is the reason behind this anomalous separation.