A nonspecific primer anchored PCR technique for chromosome walking
AUTOR(ES)
Zhang, Yan, Liu, Daqun, Yang, Wenxiang, Li, Yaning, Yan, Hongfei
FONTE
Brazilian Archives of Biology and Technology
DATA DE PUBLICAÇÃO
2011-02
RESUMO
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.
Documentos Relacionados
- T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends
- Cloning regions of the Drosophila genome by microdissection of polytene chromosome DNA and PCR with nonspecific primer.
- Ligation-anchored PCR: a simple amplification technique with single-sided specificity.
- Biologically-generated primer for PCR: PCR primer of unknown sequence.
- Screening for overlapping bacterial artificial chromosome clones by PCR analysis with an arbitrary primer