Application of AFLP® technology to radiation hybrid mapping (original) (raw)
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BioTechniques, 2000
Here, the power of the 5′ nuclease assay to detect PCR products containing (CA)n repeats was compared with that of the classical electrophoretic analysis. This assay, which relies on the use of a unique (CA)10 energy transfer-labeled probe and the 5′ nuclease activity of Taq DNA polymerase, was used to construct a dog radiation hybrid map consisting of microsatellite markers. Data from over 7000 PCRs were analyzed in parallel by the fluorogenic assay and the conventional ethidium bromide-stained, agarose gel-based assay. We show that the fluorogenic assay provides a sensitive, reliable and specific method for detecting (CA)n amplimers. Moreover, as no processing is required after the PCR, the risk of carryover contamination and the time required for sample analysis are greatly reduced. All radiation hyrid (RH) assays can be performed using a single PCR protocol, and a standard analysis method has been developed that enables numerically automated data processing. On the whole, using ...
Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs
Cytogenetics and Cell Genetics, 1998
We have developed a panel of 152 whole-genome radiation hybrids by fusing irradiated diploid pig lymphocytes or fibroblasts with recipient hamster permanent cells. The number and size of the porcine chromosome fragments retained in each hybrid clone were checked by fluorescence in situ hybridization with a SINE probe or by primed in situ labeling (PRINS) with SINE-specific primers. A strategy
Animal Genetics, 2007
High-density whole-genome maps are essential for ordering genes or markers and aid in the assembly of genome sequence. To increase the density of markers on the bovine radiation hybrid map, and hence contribute to the assembly of the bovine genome sequence, an Illumina Ò BeadStation was used to simultaneously type large numbers of markers on the Roslin-Cambridge 3000 rad bovine-hamster whole-genome radiation hybrid panel (WGRH 3000). In five multiplex reactions, 6738 sequence tagged site (STS) markers were successfully typed on the WGRH 3000 panel DNA. These STSs harboured SNPs that were developed as a result of the bovine genome sequencing initiative. Typically, the most time consuming and expensive part of creating high-density radiation hybrid (RH) maps is genotyping the markers on the RH panel with conventional approaches. Using the method described in this article, we have developed a high-density whole-genome RH map with 4690 loci and a linkage map with 2701 loci, with direct comparison to the bovine wholegenome sequence assembly (Btau_2.0) in a fraction of the time it would have taken with conventional typing and genotyping methods.
A methodological approach for the construction of a radiation hybrid map of bovine chromosome 5
Genetics and Molecular Biology, 2004
A bovine 5,000 rad WG-RH panel was used to construct an RH map of bovine chromosome 5 (BTA5). Twenty-one microsatellites and thirteen genes were scored in the panel using PAGE and radioactive labeling. Marker retention ranged from 8.9%-25.8% and averaged 17.8%. Pairwise locus analysis placed all markers in a single syntenic group with a LOD support of 4.0. At a LOD support of 8.0, a centromeric group of 23 syntenic markers was formed. Telomeric groups of 11 and 9 markers were assembled with a LOD support of 6.0 and 8.0, respectively. All markers were ordered by maximum likelihood methods using the program RHMAP. Only 13 markers were ordered with a LOD support of at least 3.0, while 25 and 29 markers were ordered with a support of at least 2.0 and 1.0, respectively. Total length of the comprehensive RH map was 435.9 cR 5,000 , with an average marker separation of 12.8 cR 5,000 . The largest gaps in the map were 55.0 and 30.4 cR 5,000 in length. The locus orders of markers common to both the RH map and the USDA-MARC linkage map were identical. The relationship between the RH and linkage maps was calculated to be 3.74 cR 5,000 /cM.
Construction of a new porcine whole-genome framework map using a radiation hybrid panel
Animal Genetics, 2003
We have constructed a radiation hybrid (RH) map of the porcine genome using an RH panel generated by an irradiation dose of 5000-rad (Sus scrofa radiation hybrid map, SSRH map). Normal porcine aortic endothelial cells were irradiated and fused with a thymidine kinasedeficient mouse cell line, L-M (TK-). A total of 110 cell lines were selected and used for further analysis. Among 1091 microsatellite (MS) markers selected for mapping, 842 markers (77%) could be typed on the panel. The framework map comprised 342 MS markers and an additional 247 MS markers were then added to generate the whole-genome map. The average retention frequency for the data set was 30.6%. The total map length was 5596.2 centiRay (cR). Using an estimated physical length of 2718 Mbp, the average ratio between cR and physical distance over the porcine genome was estimated to be 0.49 Mb/cR.
Enriching the bovine microsatellite Radiation Hybrid map with AFLP® markers
RIASSUNTO -Arricchimento della mappa RH di bovino con marcatori AFLP ® -La tecnologia AFLP è stata adattata all'analisi di pannelli di ibridi di radiazione incrementando il numero di nucleotidi selettivi al 3' dei primer utilizzati, così semplificando il profilo elettroforetico e riducendo la probabilità di comigrazione delle bande bovine con quelle di criceto. Trentasette combinazioni di primer hanno prodotto 747 bande di origine bovina. Di queste, 650 sono risultate significativamente associate con test a due punti a 1222 marcatori microsatelliti già mappati sui 29 autosomi e sui cromosomi X e Y del bovino. Si riporta la mappatura preliminare multipoint di BTA20, che conferma l'utilità dei marcatori AFLP per arricchire le mappe RH e favorire studi di clonaggio per posizione.
RIASSUNTO -Arricchimento della mappa RH di bovino con marcatori AFLP ® -La tecnologia AFLP è stata adattata all'analisi di pannelli di ibridi di radiazione incrementando il numero di nucleotidi selettivi al 3' dei primer utilizzati, così semplificando il profilo elettroforetico e riducendo la probabilità di comigrazione delle bande bovine con quelle di criceto. Trentasette combinazioni di primer hanno prodotto 747 bande di origine bovina. Di queste, 650 sono risultate significativamente associate con test a due punti a 1222 marcatori microsatelliti già mappati sui 29 autosomi e sui cromosomi X e Y del bovino. Si riporta la mappatura preliminare multipoint di BTA20, che conferma l'utilità dei marcatori AFLP per arricchire le mappe RH e favorire studi di clonaggio per posizione.
A bovine whole-genome radiation hybrid panel and outline map
Mammalian …, 2002
A 3000-rad radiation hybrid panel was constructed for cattle and used to build outline RH maps for all 29 autosomes and the X and Y chromosomes. These outline maps contain about 1200 markers, most of which are anonymous microsatellite loci. Comparisons between the RH chromosome maps, other published RH maps, and linkage maps allow regions of chromosomes that are poorly mapped or that have sparse marker coverage to be identi®ed. In some cases, mapping ambiguities can be resolved. The RH maps presented here are the starting point for mapping additional loci, in particular genes and ESTs that will allow detailed comparative maps between cattle and other species to be constructed. Radiation hybrid cell panels allow high-density genetic maps to be constructed, with the advantage over linkage mapping that markers do not need to be polymorphic. A large quantity of DNA has been prepared from the cells forming the RH panel reported here and is publicly available for mapping large numbers of loci.
BMC Genomics, 2008
Background: Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification dissociation curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and dissociation curve analysis for use as a novel scoring method.