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Single-stranded megaprimer splicing through OE-PCR: Construction of full-length <i style="">Aspergillus niger</i> arginase cDNA

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Title Single-stranded megaprimer splicing through OE-PCR: Construction of full-length Aspergillus niger arginase cDNA
 
Creator Jayashri, T N
Anuradha, R
Punekar, N S
 
Subject Single-stranded megaprimer
Overlap extension PCR
Splicing
Aspergillus niger
Arginase
 
Description 266-268
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.
 
Date 2009-06-15T12:06:48Z
2009-06-15T12:06:48Z
2009-06
 
Type Article
 
Identifier 0975-0959 (Online); 0301-1208 (Print)
http://hdl.handle.net/123456789/4590
 
Language en_US
 
Publisher CSIR
 
Source IJBB Vol.46(3) [June 2009]