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Indian Journal of Biochemistry and Biophysics (IJBB) >
IJBB Vol.46 [2009] >
IJBB Vol.46(3) [June 2009] >
| Title: | Single-stranded megaprimer splicing through OE-PCR: Construction of full-length Aspergillus niger arginase cDNA |
| Authors: | Jayashri, T N
Anuradha, R
Punekar, N S |
| Keywords: | Single-stranded megaprimer
Overlap extension PCR
Splicing
Aspergillus niger
Arginase |
| Issue Date: | Jun-2009 |
| Publisher: | CSIR |
| Abstract: | A useful variant of PCR technique was devised to generate full-length Aspergillus niger arginase cDNA for expression. Briefly, a 450 bp amplicon was first constructed through overlap extension PCR (OE-PCR) by splicing in a 101 nucleotide long single-stranded megaprimer, facilitated by inclusion of an additional, shorter forward primer in the reaction. The amplicon was suitably cloned into pBlueScript to obtain pArg440 and the insert sequenced. The full-length arginase cDNA was subsequently assembled in pArg440 and moved into pET23a for heterologous expression. An interesting feature of this strategy was not to stoichiometrically incorporate the oligonucleotide megaprimer, but use it only as an early template. This OE-PCR strategy to utilize long single-stranded megaprimer may prove handy in terms of efficiency, yield and sequence choice. |
| Page(s): | 266-268 |
| ISSN: | 0975-0959 (Online); 0301-1208 (Print) |
| Source: | IJBB Vol.46(3) [June 2009]
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