Optimization of Multiplex PCR Systems for Gene-chip Detection of Mutations in Exons in cTnI Gene Associated with FHCM

摘要

Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (FHCM). A microarray was fabricated to screen mutations in exons 3, 5, 7, and 8 in cTnI gene. Primers were designed for the PCR (polymerase chain reaction) to amplify the target DNA fragments from fresh blood samples. In order to simplify the PCR process, multiplex PCR technology was investigated in detail. The concentration of Mg~(2+) played an important role in multiplex PCR process, a properly low concentration of Mg~(2+) submitted a better speciality of PCR products. The speciality was also favored when the annealing temperature was reasonably enhanced and 64℃ is the optimal annealing temperature for the multiplex PCR systems. When applying the fabricated gene-chip to detect the target fragments from PCR mixture, the signal intensity sequence is in accordance with that from theoretic estimate.