Direct fluorescent labelling of clones by DOP PCR.
Backx L, Thoelen R, Van Esch H, Vermeesch JR.
Center for Human Genetics, University Hospital Leuven, Herestraat 49, B-3000 Leuven, Belgium. Liesbeth.backx@med.kuleuven.be.
ABSTRACT: BACKGROUND: Array Comparative Genomic Hybridisation (array CGH) is a powerful technique for the analysis of constitutional chromosomal anomalies. Chromosomal duplications or deletions detected by array CGH need subsequently to be validated by other methods. One method of validation is Fluorescence in situ Hybridisation (FISH). Traditionally, fluorophores or hapten labelling is performed by nick translation or random prime labelling of purified Bacterial Artificial Chromosome (BAC) products. However, since the array targets have been generated from Degenerate Oligonucleotide Primed (DOP) amplified BAC clones, we aimed to use these DOP amplified BAC clones as the basis of an automated FISH labelling protocol. Unfortunately, labelling of DOP amplified BAC clones by traditional labelling methods resulted in high levels of background. RESULTS: We designed an improved labelling method, by means of degenerate oligonucleotides that resulted in optimal FISH probes with low background. CONCLUSION: We generated an improved labelling method for FISH which enables the rapid generation of FISH probes without the need for isolating BAC DNA. We labelled about 900 clones with this method with a success rate of 97%.
PMID: 18471308 [PubMed - in process]PMCID: PMC2375879