Applications of interspersed repeat sequence polymerase chain reaction
- H. Zimdahl
- C. Goesele
- T. Kreitler
- M. Knoblauch
- Methods in Molecular Biology
- Methods Mol Biol 255: 113-129
Analysis of complex genomes includes characterization of complete large-insert genomic libraries comprising several hundreds of thousands of clones. Conventional methods to screen large-insert clone libraries for specific clones within a defined chromosomal interval are polymerase chain reaction (PCR) based using microsatellite markers. This strategy is labor- and cost-intensive and requires the PCR amplification of several thousands of DNA samples and verification of the PCR products via agarose gel electrophoresis. The number of PCR reactions is significantly reduced by the pooling of library clones in a three-dimensional (3D) pooling system. Nevertheless, several hundred PCR reactions are necessary to screen a P1-derived artificial chromosome (PAC) or bacterial artificial chromosome (BAC) library for one individual microsatellite marker. The application of high-density clone arrays, spotted robotically on nylon filters, offers the possibility of screening several tens of thousands of clones in a single working step. A new hybridization-based marker system has been established in the department of Hans Lehrach at the Max-Planck Institute for Molecular Genetics (Berlin, Germany). The interspersed repetitive sequence (IRS) markers are generated by amplification of genomic sequences that are located between two repetitive short interspersed repetitive elements (SINE) elements and are evenly distributed over the whole genome. IRS-PCR strategies have been applied to various species using the SINE sequences in human (Alu repeat), mouse (B1 repeat), rat (ID repeat), and zebrafish (DANA/mermaid repeat).