Recent developments in FISH technology

Recently, multicolor FISH (mFISH) and color banding have been developed. The mFISH method involves painting each of the 24 different chromosomes a different color using four or five fluorophores with combined binary ratio labeling, which allows the entire karyotype to be screened for chromosome aberrations (Figure 22.1c).21 Since the human eye cannot effectively distinguish the 24 colors, this method requires the use of an automated imaging system. In color banding, which is based on traditional banding techniques, each chromosome is labeled by subregional DNA probes in different colors, resulting in a unique chromosome bar code (Figure 22.1d).22 This method allows the rapid identification of chromosomes and chromosome rearrangements. These techniques are at present relatively new and have not been employed as widely or extensively as FISH, but their potential is high.

Of great promise in its application to FISH is rolling circle amplification (RCA), which is a molecular methodology that allows visualization by FISH of small changes previously undetectable by microscopy. It relies upon the isothermal linear amplification of a single stranded circle of DNA. A variation of RCA uses a probe with two 3' ends, a target specific sequence (40-50 nt) attached by a poly-T linker and (CH2)18 spacer to a primer (24-28 nt), which will initiate an RCA.23 When the probe is added to cells fixed onto microscope slides, the target specific sequence anchors the probe to a region of DNA and stops it from being removed during washing. Thus, when RCA occurs the product is attached to the genomic DNA; when decorator probes and biotin labeled dUTPs are added, the linear repeat can be collapsed by avidin to give a localized area of fluorescence.23 This technique means that much smaller fragments of DNA than would be apparent by traditional FISH can be visualized, including small deletions, duplications, point mutations, and even single nucleotide polymorphisms.

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