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

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dc.contributor.authorJayashri, T N
dc.contributor.authorAnuradha, R
dc.contributor.authorPunekar, N S
dc.date.accessioned2011-11-09T09:18:01Z
dc.date.available2011-11-09T09:18:01Z
dc.date.issued2009-06
dc.description.abstractA 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.en_US
dc.identifier.citationJayashri T N, Anuradha R, Punekar N S. Single-stranded megaprimer splicing through OE-PCR: Construction of full-length Aspergillus niger arginase cDNA. Indian Journal of Biochemistry & Biophysics. 2009 June; 46(3): 266-268.en_US
dc.identifier.urihttps://imsear.searo.who.int/handle/123456789/135204
dc.language.isoenen_US
dc.source.urihttps://nopr.niscair.res.in/bitstream/123456789/4590/1/IJBB%2046%283%29%20266-268.pdfen_US
dc.subjectSingle-stranded megaprimeren_US
dc.subjectOverlap extension PCRen_US
dc.subjectSplicingen_US
dc.subjectAspergillus nigeren_US
dc.subjectArginaseen_US
dc.subject.meshArginase
dc.subject.meshAspergillus niger --genetics
dc.subject.meshDNA, Complementary --metabolism
dc.subject.meshDNA primers
dc.subject.meshDNA, Single-Stranded
dc.subject.meshPolymerase Chain Reaction --methods
dc.subject.meshRNA Splicing
dc.titleSingle-stranded megaprimer splicing through OE-PCR: Construction of full-length Aspergillus niger arginase cDNA.en_US
dc.typeArticleen_US
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