Single nucleotides substitution for the formation of two-site mutagenic <i>Hggt<i/> cDNA gene in <i>Elaeis guineensis<i/> by PCR overlaps extension

Abstract

Polymerase Chain Reaction has become an easy and quick method to use in the alteration of any gene sequence, this is because single or multiple sequences changes can readily be incorporated chemically into oligonucleotide primers to produce required modification. A method of adding two mutations in Homogentisate Geranylgeranyl Transferate (HGGT) cDNA gene of Elaeis guineensis was carried out to generate three (3) different cDNA sequences molecules that are 99.99% homologous to original wild type cDNA (DxP HGGT). This is by simple and flexible method of site-directed mutagenesis driven by two step PCR overlap extension. Single nucleotide mutagenesis was successfully carried out to generate three cDNA sequence variants (193SNPHGGT, 2429SNPHGGT and 2SNPHGGT) with one or both SNP variants incorporated into the sequence of the commercial D×P genotype HGGT gene. The first PCR produces two overlapping fragments with introduced nucleotide substitution at 101 bp and 1288 bp for the first mutation, 629 bp and 760 bp for the second mutation from the full length cDNA of 1389 bp. The intermediate fragments were hybridized at their 3’end in the subsequent PCR and full length cDNA of 1389 bp were amplified by outer primers which are modified to includes gateway recombination properties to ligate the products into expression vector pB7WG2D,1 for subsequent molecular studies. Coding regions of Elaeis guineensis HGGT gene was successfully mutated by site-directed mutagenesis and amplified using PCR overlap extension without necessarily cloning the gene of interest into any plasmid before been use for mutagenesis.