You are probably familiar with fluorescent in situ hybridization (FISH) to detect and localize the presence or absence of specific DNA sequences on chromosomes. But did you know there are numerous FISH experiment variations? Including high-resolution FISH and quantitative FISH? Read here about Fiber-FISH, Q-FISH, and Flow-FISH and decide if you would like to undertake one of these methods in your laboratory.
FISH is a general technique that involves hybridizing a nucleic acid probe to its complementary sequence on chromosomes, within cells and tissues that have been previously fixed on slides. Typically in FISH the probes and targets-of-interest are visualized in situ by fluorescence microscopy.
These days, the original FISH protocol has been diversified and developed into a variety of applications, including diagnostic assays. The variation of these FISH experiments is primarily in the sequence and labeling of the probes. Today I am only going to touch on three of the more popular FISH variations: Fiber-FISH, Q-FISH, and Flow-FISH.
Fiber-FISH1 refers to FISH that is performed on extended DNA. It is a high-resolution technique that allows the mapping of genes and chromosomal regions on fibers of chromatin or DNA. In this variation, chromosomes in interphase that are stretched out in a straight line (rather than being tightly coiled, as in conventional FISH) are fixed to a microscope slide prior to hybridization. Then mechanical shearing is applied along the length of the slide to cause the extension of the DNA. This method can be performed on DNA from either cells that have been fixed to the slide and then lysed, or from a solution of purified DNA.
The benefit of using chromosomes with extended conformation is that you get dramatically higher resolution, down to a few kilobases! Therefore, fiber-FISH makes it possible to analyze epigenetic markers, such as DNA methylation and histone modification. It should be noted that only specialized laboratories routinely use the fiber-FISH technique, as it is somewhat of a skilled art. But who is to say you can’t be an artist too?
Quantitative fluorescent in situ hybridization (Q-FISH)2 is a cytogenetic-based variation of the traditional FISH technique that permits the measurement of probe signal intensity. In Q-FISH, synthetic DNA mimics are labelled with cyanine (Cy)3 or fluorescein isothiocyanate (FITC), to form peptide nucleic acid (PNA) oligonucleotides. PNA backbones contain no charged phosphate groups, therefore PNA-DNA binding is stronger than that of DNA-DNA or DNA-RNA duplexes. These PNA-conjugated probes are then used to quantify target sequences in chromosomal DNA using fluorescent microscopy and analysis software.
Q-FISH has been used mainly for measuring telomere length in vertebrates. The greatest advantage of Q-FISH over other FISH techniques is the quantitative ability of this technique. However, some disadvantages of Q-FISH include that it is very labor intensive and generally not suitable for high-throughput analysis.
Flow-FISH3 was first published in 1998 as a modification of Q-FISH. Both techniques are similar in that they analyze telomere length by employing PNA-conjugated probes to visualize and measure the length of telomere repeats. However, unlike Q-FISH, flow-FISH analysis combines in situ hybridization with flow cytometry for measurement of the telomeric signals from cells in suspension. In other words, flow-FISH can quantify median fluorescence in a population of cells, via the use of a flow cytometer, instead of a fluorescence microscope. This in turn, permits large numbers of cells to be analyzed rapidly.
All three of these FISH techniques are useful in molecular cytogenetics. They allow either visualization of genomic loci; or the ability to analyze genome organization and structure in single cells at the DNA (RNA) sequence level. Furthermore, each modified FISH technique has its own benefit versus the others. Here’s a quick re-cap:
- Fiber-FISH can obtain higher magnitudes of resolution, down to a few kilobases.
- Q-FISH is able to determine telomere length in a particular chromosome within an individual cell, whereas, flow-FISH is unable to.
- Flow-FISH can measure fluorescence intensity (and thus telomere length) in a large population of cells rather than just a handful of cells in the case of Q-FISH.
1. Ersfeld, K. (2004). Fiber-FISH: fluorescence in situ hybridization on stretched DNA. Methods Mol Biol. 270:395–402.
2. Slijepcevic, P. (2001) Telomere length measurement by Q-FISH. Methods Cell Sci. 23:17–22.
3. Rufer, N., et al. (1998) Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. 16:743–7.