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

Arbind Kumar,Jagdeep Kaur

doi:10.1155/2014/937308

Abstract

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.

Highlights

Polymerase chain reaction (PCR) based cloning of gene of interest with high GC content is a long recognized problem
The whole genome sequence of Mycobacterium tuberculosis was deciphered by Cole et al [6]
The Mycobacterium genome has very high GC content (66%) which raised the possibility of hairpin structure in the genomic structure

Summary

Introduction

Polymerase chain reaction (PCR) based cloning of gene of interest with high GC content is a long recognized problem. Presence of high GC content increased the annealing temperature beyond the extension temperature (72∘C) and repeated stretches generate the hairpin structure. In such cases, effectiveness and reproducibility of PCR amplification depend on detailed analysis of the possible secondary structures of the oligonucleotide primers as well as formation of self-dimers and cross-dimers with other interrelating oligonucleotides [7]. Effectiveness and reproducibility of PCR amplification depend on detailed analysis of the possible secondary structures of the oligonucleotide primers as well as formation of self-dimers and cross-dimers with other interrelating oligonucleotides [7] Though these problems have been considered by several investigators, no systematic details are available to approach this problem. In an attempt to clone GC rich genes (Rv0519c and Rv0774c from M. tuberculosis and ML0314c from M. leprae) from Mycobacterium sp., we designed primers by using

C C AT GGT

Materials and Methods

Results and Discussion

C T A TC

Conclusion