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

Table 1

Comparison of standard (normal) and modified primer sequences and its secondary structure for amplification of GC rich sequences.

Gene	Primers (5′-3′)	GC content	Secondary structure	Δ𝐺 (Kcal/mol)	Gene annealed at
16s DNA	F-GAG GAA GGT GGG GAT GAC GT	60	—	—	58°C
	R-AGG CCC GGG AAC GTA TTC AC	60
Rv0774c	NF-GGATCCATGATGCCCGCATGCCAGA	60		−0.92	63.5°C
	NR-AAG CTT TTA ACC TGT GAG CAG CGG CGC	55.6		−1.29
Rv0519	NF- GGATCC ATG CTA CGG CGT GGC TGTG	64		−3.67	—
	NR- AAGCTTCTAGGACGCCATCCA AGCCAG	55.6		−1.87
	MF- GGA TCC ATG CTA CGA CGA GGC TGT G	60		−0.31	64.5°C
	MR- AAGCTTCTAGGACGCCAT CCA TGC CAG	55.6		−1.25
ML0314c	NF- GAATTCTTGGCCATCAGCGCCGGAG	60		−0.97	—
	NR- AAGCTT CTA GAG GCT TTG CCT GTC TCT A	46.4		−1.45
	MF-AGAATTCTTGGCCATCAGCGCCGGAG	57.7		−0.97	62°C
	MR-GG AAG CTT CTA GAG CCT TTG CCT GTC TCT A	53.3		−1.19

NF: normal forward; NR: normal reverse; MF: modified forward; MR: modified reverse.
Data evaluation by oligo-analyzer tools on IDT: integrative DNA technology.