Primer Based Approach for PCR Amplification of High GC Content Gene: Mycobacterium Gene as a Model

The genome of Mycobacterium is rich in GC content and poses problem in amplification of some genes, especially those rich in the GC content in terminal regions, by standard/routine PCR procedures. Attempts have been made to amplify three GC rich genes of Mycobacterium sp. (Rv0519c and Rv0774c from M. tuberculosis and ML0314c from M. leprae). Out of these three genes, Rv0774c gene was amplified with normal primers under standard PCR conditions, while no amplification was observed in case of Rv0519c and ML0314c genes. In the present investigation a modified primer based approach was successfully used for amplification of GC rich sequence of Rv0519c through codon optimization without changing the native amino acid sequence. The strategy was successfully confirmed by redesigning the standard primers with similar modifications followed by amplification of ML0314c gene. 1. Introduction Polymerase chain reaction (PCR) based cloning of gene of interest with high GC content is a long recognized problem. PCR is a most sensitive tool and various factors have to be optimized for amplification of gene of interest. Primer is one of the precise control elements in this process. Designing of primers directly influences the result of standardized cloning procedures. High GC content of the gene generates complication during primer designing like mismatch and high annealing temperature, self-dimer formation, and secondary structure. Sometimes, amplification of gene is not routinely achieved by normal PCR techniques. The most prominent problem associated is hairpin loop, which directly interferes during annealing of primers on difficult DNA template that leads to no amplification. Different strategies have been proposed to sort out this problem. Use of DMSO and glycerol was reported to reduce the annealing temperature and denaturation temperature, increase the chances of breakage of secondary structure, and increase the efficiency of amplification [1–5]. The whole genome sequence of Mycobacterium tuberculosis was deciphered by Cole et al. [6]. The genes of M. tuberculosis are being cloned and expressed in E. coli cells in order to identify their possible role in Mycobacterium life. The Mycobacterium genome has very high GC content (66%) which raised the possibility of hairpin structure in the genomic structure. From genome sequence analysis it was observed that PPE, PE, and PGRS multigene family code for proteins of approximately 110–80 amino acids rich in proline and glutamic acid at N-terminal position. Proline and glutamic acid residues are mainly coded by triplet of GC