A Method for Construction, Cloning and Expression of Intron-Less Gene from Unannotated Genomic DNA |
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Authors: | Vineet Agrawal Bharti Gupta Uttam Chand Banerjee Nilanjan Roy |
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Institution: | (1) Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab, 160 062, India;(2) Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab, 160 062, India |
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Abstract: | 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. |
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Keywords: | Exon– intron Splicing SOEP MALDI Aspergillus niger |
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