A nonspecific primer anchored PCR technique for chromosome walking

A nonspecific primer anchored PCR technique for chromosome walking

A chromosome walking method was improved in this work. The new method was named as nonspecific primer anchored PCR (NPA-PCR). Nested gene specific primers were designed based on the known region and long random primer using degeneracy oligonucleotides for nonspecific anchoring. Annealing temperatures were varied to control the priming. Target sequences were obtained by PCR with random primer and gene-specific primer. Nonspecific sequence with long random primers at both ends formed stem loop structure due to inverted terminal repeats. The method was employed to isolate a gene with newly-isolated actinomycin producing strain Streptomyces setonii Z-L-22. A 0.77 kb fragment of actinomycin synthetase gene cluster was isolated from the strain. The fragments of 1474bp and 701bp were obtained, respectively, at the up and down streams of known fragment through the this method. NCBI Blast analysis showed that the walking sequence and the known sequence were located conjointly in the same cluster gene. It demonstrated that the result was correct and this technique could be useful and efficient for chromosome walking or isolating the gene.

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Main Authors: Zhang,Yan, Liu,Daqun, Yang,Wenxiang, Li,Yaning, Yan,Hongfei
Format: Digital revista
Language:English
Published: Instituto de Tecnologia do Paraná - Tecpar 2011
Online Access:http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132011000100013
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spelling oai:scielo:S1516-891320110001000132011-03-03A nonspecific primer anchored PCR technique for chromosome walkingZhang,YanLiu,DaqunYang,WenxiangLi,YaningYan,Hongfei nonspecific primer anchored PCR chromosome walking random primer A chromosome walking method was improved in this work. The new method was named as nonspecific primer anchored PCR (NPA-PCR). Nested gene specific primers were designed based on the known region and long random primer using degeneracy oligonucleotides for nonspecific anchoring. Annealing temperatures were varied to control the priming. Target sequences were obtained by PCR with random primer and gene-specific primer. Nonspecific sequence with long random primers at both ends formed stem loop structure due to inverted terminal repeats. The method was employed to isolate a gene with newly-isolated actinomycin producing strain Streptomyces setonii Z-L-22. A 0.77 kb fragment of actinomycin synthetase gene cluster was isolated from the strain. The fragments of 1474bp and 701bp were obtained, respectively, at the up and down streams of known fragment through the this method. NCBI Blast analysis showed that the walking sequence and the known sequence were located conjointly in the same cluster gene. It demonstrated that the result was correct and this technique could be useful and efficient for chromosome walking or isolating the gene.info:eu-repo/semantics/openAccessInstituto de Tecnologia do Paraná - TecparBrazilian Archives of Biology and Technology v.54 n.1 20112011-02-01info:eu-repo/semantics/articletext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132011000100013en10.1590/S1516-89132011000100013
institution SCIELO
collection OJS
country Brasil
countrycode BR
component Revista
access En linea
databasecode rev-scielo-br
tag revista
region America del Sur
libraryname SciELO
language English
format Digital
author Zhang,Yan
Liu,Daqun
Yang,Wenxiang
Li,Yaning
Yan,Hongfei
spellingShingle Zhang,Yan
Liu,Daqun
Yang,Wenxiang
Li,Yaning
Yan,Hongfei
A nonspecific primer anchored PCR technique for chromosome walking
author_facet Zhang,Yan
Liu,Daqun
Yang,Wenxiang
Li,Yaning
Yan,Hongfei
author_sort Zhang,Yan
title A nonspecific primer anchored PCR technique for chromosome walking
title_short A nonspecific primer anchored PCR technique for chromosome walking
title_full A nonspecific primer anchored PCR technique for chromosome walking
title_fullStr A nonspecific primer anchored PCR technique for chromosome walking
title_full_unstemmed A nonspecific primer anchored PCR technique for chromosome walking
title_sort nonspecific primer anchored pcr technique for chromosome walking
description A chromosome walking method was improved in this work. The new method was named as nonspecific primer anchored PCR (NPA-PCR). Nested gene specific primers were designed based on the known region and long random primer using degeneracy oligonucleotides for nonspecific anchoring. Annealing temperatures were varied to control the priming. Target sequences were obtained by PCR with random primer and gene-specific primer. Nonspecific sequence with long random primers at both ends formed stem loop structure due to inverted terminal repeats. The method was employed to isolate a gene with newly-isolated actinomycin producing strain Streptomyces setonii Z-L-22. A 0.77 kb fragment of actinomycin synthetase gene cluster was isolated from the strain. The fragments of 1474bp and 701bp were obtained, respectively, at the up and down streams of known fragment through the this method. NCBI Blast analysis showed that the walking sequence and the known sequence were located conjointly in the same cluster gene. It demonstrated that the result was correct and this technique could be useful and efficient for chromosome walking or isolating the gene.
publisher Instituto de Tecnologia do Paraná - Tecpar
publishDate 2011
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132011000100013
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