Determination of the linkage between nt 19911 and nt 20210 by simultaneous HindIII and EcoNI digestion of a 345-bp amplified fragment spanning both sites. (A) Schematic illustration of 4 possible alleles and specific fragment sizes in basepairs after cleavage at the indicated sites (X). (B) Electrophoretic separation of the following observed genotypes: 3 normal homozygotes for 20210G (lanes 1 through 3) who were GG, GA, and AA for the 19911 polymorphism, respectively; 2 homozygotes for 20210A (lanes 4 and 5) who were AA and AG for the 19911 polymorphism, respectively; and 2 heterozygotes for G20210A who were AA (lane 6) and AG (lane 7) for the 19911 polymorphism. The additional band in lane 7 between the 345- and 303-bp bands represents a heteroduplex (H.D). This was proved by the following two mixing experiments. (1) When 345-bp PCR fragments from a homozygote for 20210G and 19911G and a homozygote for 20210A and 19911A were mixed and digested with HindIII andEcoNI, an uncut 345-bp fragment and a 303-bp fragment were obtained (lane 8). (2) When DNA samples from the same 2 individuals were mixed and then amplified by PCR, followed by digestion and electrophoresis, a pattern representing heteroduplex bands was obtained (lane 9) that is identical to the one shown in lane 7. Numbers alongside the gel indicate the size (in basepairs) of the bands.