Abstract
BackgroundArray 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.ResultsWe designed an improved labelling method, by means of degenerate oligonucleotides that resulted in optimal FISH probes with low background.ConclusionWe 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%.
Highlights
Array Comparative Genomic Hybridisation is a powerful technique for the analysis of constitutional chromosomal anomalies
Since for array CGH, targets (DNA spotted on the array) have been generated from degenerate oligonucleotide primed (DOP) amplified Bacterial Artificial Chromosome (BAC) clones, we aimed to use these Degenerate Oligonucleotide Primed (DOP) amplified BAC clones as the basis of an automated Fluorescence in situ Hybridisation (FISH) labelling protocol [4,5,6,7]
DOP amplified BAC DNA was labelled by degenerate oligonucleotide-primed PCR (PCR) using either the DOP 4 primer or a mix of DOP 1, 2 and 3 primers
Summary
Array Comparative Genomic Hybridisation (array CGH) is a powerful technique for the analysis of constitutional chromosomal anomalies. 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. Since for array CGH, targets (DNA spotted on the array) 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 [4,5,6,7]. The labelling of DOP amplified BAC clones by means of either traditional labelling methods or a PCR reaction with the DOP 4 primer (see below) resulted in high levels of background We hypothesised that this background may be caused by the formation of panhandle structures of the singlestranded PCR fragments due to intramolecular re-annealing of the primers. We reasoned that small degenerate (page number not for citation purposes)
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