Abstract
BackgroundThe 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.ResultsHere, 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.ConclusionsA 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.
Highlights
The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects
The use of conventional PCR for BAC pool screening to anchor genetic markers to physical maps with a high throughput retains some shortcomings, most notably the common need to: i) isolate and normalise DNA from a large number of clones to ensure that sufficient template is available from each BAC for reliable amplification of single copy targets by conventional PCR. ii) anchor and score each marker separately. iii) use agarose gel electrophoresis as crude verification of the fidelity of target amplification. This last step is simple but comparatively slow and difficult to automate. To overcome these limitations we describe a robust method for anchoring genetic markers in physical maps based on screening two-dimensional BAC pools (Figure 1) using a combination of a modified Multiplex-Tandem PCR (MT-PCR) and High Resolution Melt analysis (HRM)
We addressed the problem of low DNA template in BAC pools using a modification of MultiplexTandem PCR (MT-PCR) (Figure 2)
Summary
The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. 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 minimise false positive and false negative results, and 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]
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