A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis

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dc.contributor.author Caligari, Peter D. S.
dc.contributor.author Wilkinson, Michael J.
dc.contributor.author Vu, Giang T. H.
dc.date.accessioned 2011-06-01T09:11:48Z
dc.date.available 2011-06-01T09:11:48Z
dc.date.issued 2010-05-12
dc.identifier.citation Caligari , P D S , Wilkinson , M J & Vu , G T H 2010 , ' A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis ' BMC Genomics , vol 11 , no. 301 , pp. paper 301 . en
dc.identifier.issn 1471-2164
dc.identifier.other PURE: 164590
dc.identifier.other dspace: 2160/6859
dc.identifier.uri http://hdl.handle.net/2160/6859
dc.identifier.uri http://www.biomedcentral.com/1471-2164/11/301 en
dc.description Vu, G.T.H., Caligari, P.D.S., Wilkinson, M.J. (2010). A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis.   BMC Genomics, 11, paper 301. IMPF: 04.20 Sponsorship: Biohybrids International Ltd /Sumatra Biosciences en
dc.description.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. en
dc.language.iso eng
dc.relation.ispartof BMC Genomics en
dc.title A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis en
dc.type Text en
dc.type.publicationtype Article (Journal) en
dc.identifier.doi http://dx.doi.org/10.1186/1471-2164-11-301
dc.contributor.institution Institute of Biological, Environmental and Rural Sciences en
dc.description.status Peer reviewed en


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