![]() High-throughput syntheses of oligonucleotides were also easily achieved by DNA synthesis machines ( Rayner et al., 1998 Cheng et al., 2002 Livesay et al., 2002 Pon & Yu, 2004, 2005). ![]() ![]() PCR is a key component in most of the recently developed methods for gene synthesis including thermodynamically balanced inside-out (TBIO) ( Gao et al., 2003), two-step total gene synthesis coupling with dual asymmetrical PCR and overlap extension PCR ( Young & Dong, 2004), PCR-based two-step DNA synthesis (PTDS) ( Xiong et al., 2004a), successive extension PCR ( Xiong et al., 2004b, 2006a) and microchip-based technology for multiplex gene synthesis ( Tian et al., 2004). These methods included self-priming PCR ( Dillon & Rosen, 1990 Ciccarelli et al., 1991 Prodromou & Pearl, 1992), dual asymmetrical PCR (DA-PCR) ( Sandhu et al., 1992), PCR-based assembly ( Stemmer et al., 1995) and the template-directed ligation (TDL) ( Strizhov et al., 1996). In the 1990s, PCR-based strategies were used to improve chemical synthesis and assembly of DNA molecules. However, the lengths of the early synthesized DNA sequences using these techniques were generally <1.0 kb ( Beattie et al., 1988 Engels & Uhlmann, 1988). Oligonucleotides were assembled into functional genes through enzymatic ligation ( Smith et al., 1982 Edge et al., 1983 Jay et al., 1984 Sproat & Gait, 1985) or the FokI method ( Mandecki & Bolling, 1988). The efficiency of chemical synthesis of DNA sequences was further improved when synthesis of more than 100-nt long oligonucleotides became possible in the mid 1980s ( Caruthers et al., 1985). These studies have also demonstrated that chemical DNA synthesis methods are capable of making biologically functional genes ( Itakura & Riggs, 1980). Then, a large number of protein encoding genes were chemically synthesized and expressed in Escherichia coli in the 1970s and early 1980s ( Goeddel et al., 1979 Tanaka et al., 1982 Ohsuye et al., 1983). In addition, the first protein-encoding gene, somatostatin, was chemically synthesized in 1977 ( Itakura et al., 1977). In 1976, two small genes, one coding a regulatory lac operator and another a tyrosine suppressor, were chemically synthesized and cloned ( Heyneker et al., 1976 Kleppe et al., 1976). tRNA genes, could be tracked back to the Khorana group as early as the 1960s ( Gupta et al., 1968a, b Kleppe et al., 1976). The earliest enzymatic gene synthesis, e.g. A few organic chemists, foremost among them HG Khorana, developed one of the first sets of technologies to synthesize oligonucleotides in the 1960s and early 1970s. The design and construction of synthetic genes were a dream of many scientists 30 years ago. In addition, chemical gene synthesis may be preferable to avoid tedious and costly site-directed mutagenesis and subcloning ( Gao et al., 2003 Shevchuk et al., 2004 Xiong et al., 2004a). Furthermore, characterization of gene function often requires expression in a heterologous system ( Daly & Hearn, 2005 Jana & Deb, 2005 Macauley et al., 2005 Peng et al., 2006a), and in these cases, codon optimization is often necessary in order to achieve a high level of expression ( Sharp et al., 1986 Murray et al., 1989 Kurland, 1991 Kane et al., 1995 Xiong et al., 2005, 2006a). In many cases, chemical synthesis may be the only choice because template DNAs are not readily available. Chemical gene synthesis, PCR, primer design, error correction, application, biotechnology IntroductionĬhemical synthesis of DNA sequences offers a highly effective technique to elucidate gene functions and analyze protein–nucleic acid interactions ( Beattie et al., 1988 Engels & Uhlmann, 1988).
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