Identification of a mutation in the CMMB by multiplexed RT-PCR and TGCE, followed by DNA sequencing to identify the specific base pair substitution. (A) Agarose gel electrophoresis of multiplexed RT-PCR reactions. Shown are four representative samples from each of two independent three-fold multiplexed PCR reactions (multiplex 7 and 10) of cDNA templates in the CMMB. Sizes (bp) of molecular weight markers are shown on the left. Sizes (bp) of RT-PCR products of the multiplex 10 reaction are shown on the right. The asterisk indicates the product (510 bp) in which a mutation was identified by TGCE in panel B. (B) TGCE electropherogram profiles of three-fold multiplexed PCR products (multiplex 10) derived from CMMB mouse #131 (top) and C57BL/6J control (bottom) cDNA templates. A mutation (heteroduplex, red arrow) was identified in CMMB #131 in a 510-bp product derived from the Ap2a1 gene. (C) The mutation was confirmed by repeating the RT-PCR and TGCE analysis of only the 510-bp product amplified from the CMMB #131 cDNA sample. (D) DNA sequence analysis of the 510-bp products amplified from the CMMB #131 and control cDNAs revealed an A-to-G nucleotide substitution (red arrow) in the Ap2a1 gene in the CMMB #131 sample, which causes a Glu414Gly amino acid substitution in the encoded protein.