Genescan analysis software

Approximately 1 µL of the PCR product was mixed with 24 µL of formamide and 1 µL of the GeneScan 500 Rox size standard (Applied Biosystems). The mixture was denatured at 95°C for 3 minutes and placed on ice to prevent re-annealing until further analysis. The electrophoretic analysis was performed using a POP4 gel (Applied Biosystems) on the ABI 3130×l Genetic Analyzer (Applied Biosystems). The PCR products were separated and visualized using GeneScan Analysis software (Applied Biosystems). GeneMapper® ID Software v3.2 (Applied Biosystems) was used for the data analysis. The relative probe signal ratios were calculated based on the peak area of the segmental duplication (length). The expected value for a euploid sample is 1, and the expected value for a trisomic sample is 1.5, reflecting the additional target chromosome (Figure 1C).

The 5′-flanking region of the HO-1 gene containing GT-repeats was amplified by polymerase chain reaction (PCR) with a FAM-labeled sense primer (5′-AGAGCCTGCAGCTTCTCAGA-3′) and an antisense primer (5′-ACAAAGTCTGGCCATAGGAC-3′) according to a published protocol [7] (link). The PCR products were mixed with the GenoType TAMRA DNA Ladder (size range: 50–500 bp; Invitrogen, Grand Island, NY) and analyzed in an automated DNA sequencer (ABI Prism 377, Foster City, CA). The respective sizes of the GT-repeats were calculated using GeneScan Analysis software (PE Applied Biosystems, Foster City, CA). To further confirm the sizes of the GT-repeats, 3 PCR products were subcloned into the pCRII vector (Invitrogen) and subjected to the sequence analysis. For quality control purposes, approximately 10% of the samples were re-genotyped in a blinded fashion, and from which the same results were obtained.