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Oligonucleotide Fluorescent In Situ Hybridization Assay

IP.com Disclosure Number: IPCOM000180392D
Publication Date: 2009-Mar-09

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OLIGONUCLEOTIDE FLUORESCENT IN SITU HYBRIDIZATION ASSAY

The present concept relates in general to fluorescent in situ hybridization assay and, in particular, to methods for designing probes for fluorescent in situ hybridization assay.

Fluorescent in situ hybridization (FISH) is one of the commonly used cytogenetic techniques for detecting specific DNA sequences on chromosomes. The detection includes hybridizing fluorescent DNA probes with the chromosomes that contains target genomic DNA and then visualizing the position of hybridized DNA within each chromosome by using fluorescence microscopy. Accurate FISH results are obtained when the hybridization efficiency of the probes and the intensity of the fluorescent signals received are high. Fluorescent probes are generated from a template DNA using nick translation or random priming methods with fluorescent nucleotides. Traditionally, large templates of DNA, usually greater than 100 kilo base pairs (kbp), such as bacterial artificial chromosomes (BACs) are used to generate probes for FISH. However, the use of BACs is a limiting factor for FISH analysis since BACs do not resolve small-scale changes in the target genomic DNA. The generation of BACs involves a random selection process that may not cover the sequences present at the gene boundaries. Further, the use of BACs does not specifically exclude non-optimal regions such as repetitive regions, non-unique regions, hairpin susceptible sequences, and so forth. Conversely, the use of small templates such as plasmids or polymerase chain reaction (PCR) products for detecting small target genomic DNA may generate low intensity fluorescent signals. The low intensity fluorescent signals result in low signal-to-noise ratio, since the low intensity fluorescent signals are difficult to distinguish from the fluorescent signals generated due to the background noise, which may be a result of non-specific hybridization.

The present concept provides a method for designing oligonucleotide FISH probes using a duo-probe approach. The unique duo-probe approach includes use of two types of probes—genome probes and signal probes. The genome probes hybridize to a target genomic DNA. The signal probes bind to the ends of the genomic probes and generate fluorescent signals that indicate hybridization of the genome probes to the target genomic DNA, and detection of the targeted genomic sequence. The oligonucleotide FISH probes are designed in a manner such that the non-optimal regions are specifically excluded. Signal intensities obtained from small target genomic DNA can be further amplified by adding multiple signal probe layers to generate high-intensity fluorescent signals with increased signal-to-noise ratio. Thus, the present concept provides a method for designing oligonucleotide FISH probes that facilitate efficient detection...