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The study nucleoti de substitution is very important both to our understanding of gene evolution and to reliable estimation of phylogenetic relationships. In this paper nucleotide substitution is assumed to be random and the Markov model is applied to the study of the evolution of genes. Then a non-linear optimization approach is proposed for estimating substitution in real sequences. This substitution on is called the Nucleotide State Transfer Matrix. One of the most important conclusions from this work is that gene sequence evolution conforms to the Markov process. Also, some theoretical evidences for random evolution are given from energy analysis of DNA replication.