Restriction Site Extension PCR: A Novel Method for High-Throughput Characterization of Tagged DNA Fragments and Genome Walking

BACKGROUND: Insertion mutant isolation and characterization are extremely valuable for linking genes to physiological function. Once an insertion mutant phenotype is identified, the challenge is to isolate the responsible gene. Multiple strategies have been employed to isolate unknown genomic DNA that flanks mutagenic insertions, however, all these methods suffer from limitations due to inefficient ligation steps, inclusion of restriction sites within the target DNA, and non-specific product generation. These limitations become close to insurmountable when the goal is to identify insertion sites in a high throughput manner. METHODOLOGY/PRINCIPAL FINDINGS: We designed a novel strategy called Restriction Site Extension PCR (RSE-PCR) to efficiently conduct large-scale isolation of unknown genomic DNA fragments linked to DNA insertions. The strategy is a modified adaptor-mediated PCR without ligation. An adapter, with complementarity to the 3' overhang of the endonuclease (KpnI, NsiI, PstI, or SacI) restricted DNA fragments, extends the 3' end of the DNA fragments in the first cycle of the primary RSE-PCR. During subsequent PCR cycles and a second semi-nested PCR (secondary RSE-PCR), touchdown and two-step PCR are combined to increase the amplification specificity of target fragments. The efficiency and specificity was demonstrated in our characterization of 37 tex mutants of Arabidopsis. All the steps of RSE-PCR can be executed in a 96 well PCR plate. Finally, RSE-PCR serves as a successful alternative to Genome Walker as demonstrated by gene isolation from maize, a plant with a more complex genome than Arabidopsis. CONCLUSIONS/SIGNIFICANCE: RSE-PCR has high potential application in identifying tagged (T-DNA or transposon) sequence or walking from known DNA toward unknown regions in large-genome plants, with likely application in other organisms as well

Molecular analysis of chronic granulomatous disease and site-direted mutagenesis as models to solve the naden oxidase puzzle

IP 1115-04-476-96ARTICULO(S) EN REVISTA: Recombination events between the p47 phox gene andits highly homologous pseudogenes;are the main cause of autosomal recessive chronic granulomatusdisease / Joachim Roesler ... [et al.]. -- En:;Blood. -- Vol.95, no. 6 (Mar. 2000); p. 2150-2156. -- Molecularcharacterization of autosomal recessive;chronic granulomatous disease caused by a defect of the nicotinamide adenine dinucleotide phosphate (reduced;form) oxidase component p67 phox / Pablo J. Patiño ... [etal.].'-- en: Blood. -- Vol. 94, no. 7 (1999); p.;2505-2514. -- Molecular analysis of chronic granulomatousdisease caused by defects in gp91 phox / Pablo J.;Patiño ... [et al.]. -- En: Human Mutation. -- no. 13 (1999);p.29-37. --Caracterizacion clinico molecular;de la enfermedad granulomatosa cronica autosomica recesivacausada por deficit de p47-phox / Monica Cornejo de;L. --- [et al.]. -- En: Revista Medica de Chile. -- Vol. 128 (2000); p.490-498