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Open Access Highly Accessed Methodology article

Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

Zhen Wang, Shafei Ye, Jingjing Li, Bo Zheng, Manzhu Bao and Guogui Ning*

Author Affiliations

Key laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, P. R. China

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BMC Biotechnology 2011, 11:109  doi:10.1186/1472-6750-11-109

Published: 17 November 2011

Additional files

Additional file 1:

Tables for listing different used primers. Table S1. AD primer (arbitrary degenerate primers) and universal primers used in this study. Table S2. FP primers (exhibiting restriction site) and the corresponding universal primers used in FPNI-PCR. Table S3. FP primers (exhibiting hair pin structure) and the corresponding universal primers used in FPNI-PCR. Table S4. Gene specific primers used to generate the electrophoresis patterns presented in this paper. Table S5: Gene specific primers used to generate the electrophoresis patterns presented when conducting genomic walking in Arabidopsis and rice.

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Additional file 2:

Supporting figures employed in main text. Figure S1. Illustration of the effect of annealing temperature during the low stringency PCR cycles in the primary PCR step in FT ortholog cloning of Fragaria ananassa using FPNI-PCR (amplified products after the secondary round of PCR). M: molecular marker; number in the lanes: 1-9 FP primer; number in the bottom right corner: annealing temperature. -: control. Figure S2. Amplified products after the tertiary round of PCR in genomic walking and T-DNA flanking sequence cloning using FPNI-PCR for the 3 genes of wuschel family from Arabidopsis (a), and Osft, Osmads1 and Ostuba1 genes from rice (b) (FP primer indicated by number in photos).

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