Molecular cloning of human cardiac troponin I using polymerase chain reaction

Abstract

We have used the polymerase chain reaction (PCR) to synthesise a cDNA encoding part of human cardiac troponin I. Amplification was achieved using fully degenerate sets of oligonucleotides corresponding to conserved regions of amino acid sequence identified in other troponin I isoforms. The cloned PCR fragment was subsequently used to isolate full-length cDNAs from a cardiac cDNA library. We describe the approach, as a general cloning strategy starting from limited amino-acid sequence data and report the cloning, and complete amino acid sequence of human cardiac troponin I. Analysis of human development using these clones demonstrates early expression of this gene in the heart.