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. 2014:2014:937308.
doi: 10.1155/2014/937308. Epub 2014 Mar 24.

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

Arbind Kumar  1 , Jagdeep Kaur  1
Affiliations

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

Arbind Kumar et al. Mol Biol Int. 2014.

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.

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Figures

Figure 1
Figure 1
Prediction of secondary structure. (a) Presence of secondary structures in the primers designed by standard primer designing. Secondary structure halts the progression of polymerase. (b) Modification of bases at wobble position distorts secondary structure and allows moving of polymerase. Base modifications are shown in light red circle and the possible hydrogen bond is shown in square box.
Figure 2
Figure 2
Amplification pattern of 16S rDNA, Rv0519, and Rv0774c genes from M. tuberculosis (Figures 2(a) and 2(b)), ML0314c and 16S rDNA from M. leprae (Figures 2(c) and 2(d)). (a) Amplification of 16S rDNA, Rv0519, Rv0774c genes with standard primers, (b) amplification of Rv0519 with modified primers, (c) amplification of 16S rDNA and ML0314c genes with standard primers, and (d) amplification of ML0314c with modified primers.
Figure 3
Figure 3
Schematic presentation of the annealing primer mismatch and use of wobble hypothesis approach to sort out this problem.
See this image and copyright information in PMC

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