Design of primers and optimization of PCR conditions for the detection of alternatively spliced isoforms of mouse ChAT mRNA

Abstract

Acetylcholine plays an important role as a neurotransmitter in the central and peripheral nervous systems. Its biosynthesis is catalyzed by choline acetyltransferase (ChAT). In mice, the ChAT gene has three 5′-noncoding (R, N, and M) and 14 coding exons, from which seven mRNA isoforms (M, N1, N2, R1, R2, R3, and R4) are transcribed. They differ in the 5′-noncoding ends and encode the same protein (common ChAT). An additional isoform that lacks coding exons from 5 to 8 encodes a smaller protein (peripheral ChAT). This research aimed to design and check the specificity of primers targeting R3, N1, N2, M, peripheral ChAT, and common ChAT isoforms. The optimal PCR conditions and specificity of the primers were tested in a series of PCR reactions. Specially designed DNA fragments or plasmids containing isoform-specific nucleotide sequences were used as templates. The appropriate annealing temperature, which yields sufficient specificity for each tested primer pair, was as follows: R3 – 60 °C; N1 – 63 °C; N2 – 65 °C; M – 65 °C; peripheral ChAT – 65 °C and common ChAT – 63 °C.