Abstract
Oligonucleotides fluorescence in situ hybridization (Oligo-FISH) is an emerging technology and is an important tool in research areas such as detection of chromosome variation, identification of allopolyploid, and deciphering of three-dimensional (3D) genome structures. Based on the demand for highly efficient oligo probes for oligo-FISH experiments, increasing numbers of tools have been developed for probe design in recent years. Obsolete oligonucleotide design tools have been adapted for oligo-FISH probe design because of their similar considerations. With the development of DNA sequencing and large-scale synthesis, novel tools have been designed to increase the specificity of designed oligo probes and enable genome-scale oligo probe design, which has greatly improved the application of single copy oligo-FISH. Despite this, few studies have introduced the development of the oligo-FISH probe design tools and their application in FISH experiments systematically. Besides, a comprehensive comparison and evaluation is lacking for the available tools. In this review, we provide an overview of the oligo-FISH probe design process, summarize the development and application of the available tools, evaluate several state-of-art tools, and eventually provide guidance for single copy oligo-FISH probe design.
Highlights
Fluorescent in situ hybridization (FISH) is a powerful technique to detect and locate a specific DNA sequence on a chromosome
As we discussed above, specificity is the key consideration for a successful oligo-FISH experiment, the specificity of the probes designed by these tools needs to be inspected
The results showed that the iFISH-40 mer dataset had a higher density than the OligoMiner Balance (OMB) dataset, which indicates that the iFISH dataset is more suited for designing oligo-FISH probes in the human genome, especially for small regions
Summary
Fluorescent in situ hybridization (FISH) is a powerful technique to detect and locate a specific DNA sequence on a chromosome. With the development of DNA sequencing and synthesis technologies, a new generation of FISH techniques, called oligonucleotide fluorescence in situ hybridization (oligo-FISH), which utilizes single-copy oligonucleotides (oligos) as FISH probes, has been established. Oligo-FISH probes offer many advantages compared with conventional probes derived from ribosomal DNA (rDNA), bacterial artificial chromosome (BAC) sequences, or repetitive sequences. RDNA and many other tandemly repeated sequences can generate strong FISH signals, they are not suitable for comparative cytogenetic studies because of their prevalence and diversity among different species [9,10,11]. Synthetic oligo probes derived from a single copy region of genome allow for hybridization to precisely defined targets without the interference of repetitive sequences, and are easy to maintain
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