Vu et al. BMC Genomics 2010, 11:301 http://www.biomedcentral.com/1471-2164/11/301 M E T H O D O L O G Y A R T I C L E Open Access MAetshoimdolopgyleart,ichle igh throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis Giang TH Vu1, Peter DS Caligari2,3 and Mike J Wilkinson*1 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. Background Whole genome sequence data is currently unavailable for the overwhelming majority of plant species, including most crops. For such cases, the integration of linkage and physical maps provides a vital information platform to accelerate processes such as positional cloning, compara- tive genome analysis, and clone-by-clone sequencing [1- 3]. One key limiting step in compiling a comprehensive link between physical and linkage maps is the ability to locate large insert clones (e.g. from Bacterial Artificial * Correspondence: jjw@aber.ac.uk 1 Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Penglais, Aberystwyth, Ceredigion, SY23 3DA, UK Full list of author information is available at the end of the article Chromosome [BAC] or Yeast Artificial Chromosome [YAC] libraries) that contain the polymorphic markers used in linkage mapping. Traditionally, this has been achieved by colony hybridisation using (usually) radioac- tively-labelled cloned DNA, PCR products, or oligonucle- otides [4,5]. There are several disadvantages of using colony hybridisation as a means of identifying clones that contain target DNA; these largely centre on the difficulty in setting appropriate hybridization conditions to mini- mise false positive and false negative results, but also include the need for appropriate facilities and procedures to handle radio-labelled probes [6]. These problems can be overcome if a PCR-based screening strategy is adopted using Sequence Tagged Site (STS) markers [6,7]. © 2010 Vu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attri- BioMed Central bution 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.