Synthesis of the DNA probe for the determination of rat AChE mRNA.
This is a preliminary report on our attempts of synthesis by polymerase chain reaction (PCR), the cDNA probe for the determination of mRNA of the AChE catalytic subunit. As our strategy we took the advantage of the fact that sequence identity of AChE gene increases with phylogenetic proximity. Single codon usage could therefore be applied. Two non-degenerate PCR primers were synthesised corresponding to AChE regions which were highly conservative among species analyzed until now. The sequence amplified by these two primers should be 339 base pairs long as concluded from mouse AChE sequence. By determining the nucleotide sequence of the PCR product and by comparison of this sequence with the corresponding mouse AChE region, we would be able to verify the correspondence of our PCR product to the rat AChE gene fragment. Only the first four amino acids of our PCR product flanking Phe 200, which is the first amino acid from the A2 primer, are 100% homologous with the mouse AChE. However, from the next 18 amino acids towards the N-terminal, only 4 are homologous with the mouse AChE. Since we expected more than 90% homology between the phylogenetically closely related species of mouse and rat, we doubt that the DNA sequence obtained belongs to the rat AChE gene.
Grubic
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Institute of Pathophysiology, School of Medicine, University of Ljubljana, Slovenia.
Komel
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D000110:Acetylcholinesterase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015342:DNA Probes / Q000138:chemical synthesis*; D005784:Gene Amplification; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D017386:Sequence Homology, Amino Acid
D015342:DNA Probes; D012333:RNA, Messenger; D000110:Acetylcholinesterase