Please use this identifier to cite or link to this item:
https://hdl.handle.net/2440/69207
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Type: | Journal article |
Title: | A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis |
Author: | Vu, G. Caligari, P. Wilkinson, M. |
Citation: | BMC Genomics, 2010; 11(301):1-8 |
Publisher: | BioMed Central Ltd. |
Issue Date: | 2010 |
ISSN: | 1471-2164 1471-2164 |
Statement of Responsibility: | Giang TH Vu, Peter DS Caligari and Mike J Wilkinson |
Abstract: | Background: The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR. Results: Here, we report the first combined use of Multiplex Tandem PCR (MT-PCR) and High Resolution Melt (HRM) analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker. Conclusions: A pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes. |
Keywords: | Chromosomes, Artificial, Bacterial Arabidopsis Chromosome Mapping Gene Library |
Description: | Extent: 8p. |
Rights: | © 2010 Vu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
DOI: | 10.1186/1471-2164-11-301 |
Published version: | http://dx.doi.org/10.1186/1471-2164-11-301 |
Appears in Collections: | Agriculture, Food and Wine publications Aurora harvest |
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hdl_69207.pdf | Published version | 1.26 MB | Adobe PDF | View/Open |
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