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Single-stranded megaprimer splicing through OE-PCR: Construction of full-length Aspergillus niger arginase cDNA

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Title Single-stranded megaprimer splicing through OE-PCR: Construction of full-length Aspergillus niger arginase cDNA
 
Creator JAYASHRI, TN
ANURADHA, R
PUNEKAR, NS
 
Subject overlap extension pcr
multiple-site mutagenesis
protocol
dna
single-stranded megaprimer
overlap extension pcr
splicing
aspergillus niger
arginase
 
Description 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.
 
Publisher NATL INST SCIENCE COMMUNICATION
 
Date 2011-08-19T06:46:28Z
2011-12-26T12:56:06Z
2011-12-27T05:44:17Z
2011-08-19T06:46:28Z
2011-12-26T12:56:06Z
2011-12-27T05:44:17Z
2009
 
Type Article
 
Identifier INDIAN JOURNAL OF BIOCHEMISTRY & BIOPHYSICS, 46(3), 266-268
0301-1208
http://dspace.library.iitb.ac.in/xmlui/handle/10054/10260
http://hdl.handle.net/10054/10260
 
Language en