Schematic diagram of the HUMARA assay. Maternal and paternal X chromosomes carry different numbers of CAG repeats at the Humara locus. HpaII methylation sensitive sites are located at the polyorphic CAG region. During embriogenesis, random X chromosome inactivation occurs in female individuals, resulting in methylation of either the paternal or maternal X chromosome in different cells. Therefore, a monoclonal cell population, derived from the division of a single ancestor cell, shares the same inactivated X chromosome, whereas a polyclonal population, derived from more than one ancestor cell, may contain cells with inactive maternal and paternal X chromosomes. HpaII digestion removes the unmethylated alleles, allowing amplification of the methylated HUMARA locus. Electrophoresis of the PCR products will indicate monoclonal or polyclonal cell populations, as a single band or two bands of different size, respectively. HpaII: denotes the methylation sensitive endonuclease sites; arrows: indicate primer annealing regions; cross bars: indicate the methylated chromosome.