Clonality detection by the HUMARA assay. (A) Methylation of the inactive X chromosome at the HUMARA locus occurs early in embryonic development. Shaded, inactivated X chromosome. (B) Schematic of HUMARA locus. Crossbar indicates methylation sensitive endonuclease site; M, methylated methylation sensitive endonuclease site; CAG-p, paternal CAG tandem repeat sequence; CAG-m, maternal CAG tandem repeat sequence. (C) After digestion by methylation sensitive endonucleases, only inactive (methylated) DNA remains intact. (D) Only intact DNA can be amplified by polymerase chain reaction with flanking primers. Products can be differentiated from each other by the number of tandem CAG repeats in CAG-p and CAG-m and their resultant different electrophoretic mobility.