A method for construction, cloning and expression of intron-less gene from unannotated genomic DNA.
The present century has witnessed an unprecedented rise in genome sequences owing to various genome-sequencing programs. However, the same has not been replicated with cDNA or expressed sequence tags (ESTs). Hence, prediction of protein coding sequence of genes from this enormous collection of genomic sequences presents a significant challenge. While robust high throughput methods of cloning and expression could be used to meet protein requirements, lack of intron information creates a bottleneck. Computational programs designed for recognizing intron-exon boundaries for a particular organism or group of organisms have their own limitations. Keeping this in view, we describe here a method for construction of intron-less gene from genomic DNA in the absence of cDNA/EST information and organism-specific gene prediction program. The method outlined is a sequential application of bioinformatics to predict correct intron-exon boundaries and splicing by overlap extension PCR for spliced gene synthesis. The gene construct so obtained can then be cloned for protein expression. The method is simple and can be used for any eukaryotic gene expression.
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab 160 062, India.
Vineet
V
Agrawal
Bharti
B
Gupta
Uttam Chand
UC
Banerjee
Nilanjan
N
Roy
D010832:6-Phytase / Q000235:genetics; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004271:DNA, Fungal / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D018745:Genome, Plant / Q000235:genetics*; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D012984:Software; D014170:Transformation, Genetic
D004271:DNA, Fungal; D010832:6-Phytase