Bisulfite PCR cloning and sequencing to validate methylation status of an unmethylated Alu insertion (chr9:37594172-37594310). Asterisk indicates the CpG dinucleotides that are flanking the Alu element; the scheme shows the relative location of TOMM5 and the CpG island in relation to the Alu AI19 insertion (USCS Genome Bioinformatics). a) methylation status of a downstream AluJo (sequence coordinates: chr9:37594745-37595002) near the newly inserted Alu element; b) newly inserted Alu element and its methylation status; c) methylation status of 2 CpGs upstream of the newly inserted Alu; d), e), f), and g) methylation statuses of 3 Alu repeats and 1 MIR element localized between the newly inserted Alu and the CpG island, respectively, AluSx, AluJo, MIRb, and AluSx; h) methylation status of the 5'end of a CpG island located 1,576 bp (sequence coordinates: chr9:37592324-37592701) upstream from the newly inserted Alu element. Note that the TOMM5 transcription unit is in opposite orientation to that of the newly inserted Alu element. The methylation levels for a, b, c, d, e, f, g, and h were 40%, 33.7%, 79.1%, 4.2%, 0%, 3%, 32%, and 0.6%, respectively.