Genetic analysis of the mutational insertion at exon 8 of the PKLR gene. (A) PKLR DNA sequence analysis of healthy exon 8 compared with patient exon 8. The homozygous 11-bp insertion site at the propositus exon 8 and the oligonucleotide primer used in multiplex RT-PCR are indicated. (B) Multiplex RT-PCR amplification using the specific insertion primer. Three different fragments were obtained: a single 284-bp fragment from the normal sequence (lane 1) and 2 mutant fragments (217 bp and 295 bp) from the insertional allele. Diagram below shows the multiplex assay. The patient (lane 2) is homozygous for the mutant allele. Her mother (lane 3) and father (lane 4) are heterozygous for the wild-type and mutant alleles.