Rapid and simultaneous multiple genotyping of human Y chromosome from a human blood sample by on-chip enzymatic digestion and microchannel array electrophoresis coupled with blood sample pretreatment, and microscale PCR

Abstract

In this paper, with distinct six DNA markers, 47z, SRY, YAP, 12f2, TB4Y and UTY, as the judgement standard, a combined technique for the rapid haplotyping (multiple genotyping) of human Y chromosome was developed by a DNA analysis system. By Generation capture disk, 5 min were enough to extract and purify DNA from five samples with only 4.5 L blood for each one. Consequently, LightCycler was chosen for the amplification of the six fragments of each sample. Under the optimized conditions, the simutaneous amplification of DNA extracted from five samples could be finished within 25 min. Among the six genomic fragments of each sample, the polymorphisms of YAP and 12f2 could be distinguished directly from PCR products. The other four were single nucleotide polymorphisms. Therefore, microchip-based restriction fragment length polymorphism analysis was carried out. With a 12-channel microchip, in 8 min, two blood samples could be analyzed at the same time through on-chip digestion and the subsequent separation of DNA fragments in the channel. In summary, with the above-mentioned technique, microscale haplotyping of human Y chromosome could be finished at the average speed of 10 min/sample with only 4.5 μL blood.