Direct fluorescent labelling of clones by DOP PCR

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%.

• Publication year

  2008

• Venue

  Molecular Cytogenetics

• Publication date

  2008-03-26

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1186/1755-8166-1-3PMID 18471308PMCID 2375879
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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