previous page PLNT3140 Introductory Cytogenetics
Lecture 5, part 3 of 3
first page

II. Fluorescence In-situ Hybridization (FISH)

Fluorescence In-situ hybridization (FISH) uses fluorescence to highlight the site of specific DNA sequences. Trask et al. 1993 provide a detailed review of the technique and its applications. In essence, a DNA sequence is 'tagged' by incorporating fluorescently-labeled nucleotides into the DNA chain. The tagged DNA, referred to as a  probe, is incubated with chromosomes on a slide. Wherever the chromosome contains the same sequence as the probe, the probe can base-pair with the chromosome. The site to which the probe base pairs will fluoresce in UV microscopy.

FISH is sensitive enough to detect a single copy gene on a chromosome.

As with Geimsa banding, chromosomes must be fixed before denaturation. It is necessary to denature both the DNA probe and the target chromosome to allow complementary pairing to take place.

Fixative: The chromosomes are fixed in a 3:1 methanol:acetic acid solution. Slides are prepared by the squash method and the cover glass is removed to allow the cells to dry. The squash method stabilizes the chromosomes, permits good penetration of the reagents and brings most of the chromosome into the same focal plane, and also increases the speed at which the hybridization takes place, by decreasing volume.

Denaturation : incubate at 72 C in 50-70% formamide and 2X SSC (NaCl) (formamide denatures DNA helices). DNA remains covalently attached to nuclear matrix proteins only at Matrix Attachment Regions (MAR). Each loop of DNA between MAR sites is referred to as a chromatin domain. All other parts of the DNA helix are now single-stranded.

Annealing of probes and renaturation: The probe molecules are fragments of DNA, selected to target specific sequences on the chromosome. The target sequences can be as small as 1kbp or larger than 15 kbp. Sequences can be taken from entire chromosomes to highlight large regions of the genome.

Denatured probe is hybridized with fixed chromosomes under a coverslip overnight at 37 C in 50% formamide, 2X SSC and 10% detran sulfate. After incubation, unannealed probe is washed off . Because the probe is present in a large molar excess, compared to the chromosomal DNA, the probe will outcompete the single-stranded genomic DNA when base pairing with the strand complementary to the probe. Re-annealed duplexes are a hybrid of probe and native chromosomal DNA strands.

In summary, the chromosomes have been exposed to methanol, acetic acid, flattening, drying, rehydration, heat, formamide, and high salt concentrations during the FISH procedure. Some degree of structural changes will have been produced in the chromosomes. High resolution microscopic equipment is required for FISH as well as image processing software to enhance and analyse the image.

FISH is being used to study genome organization, the distribution of chromatin in interphase nuclei, and chromosome abnormalities. Greater resolution is possible in interphase chromosomes due to the dispersed state of the chromatin, so that small structural changes can be detected. The compaction of metaphase chromosomes sets the limit of detection at sequences separated by more than 1 Mbp. The limit in interphase chromosomes is a separation of 100 kbp (Figure 8, Trask et al. 1993).

Detection of single genes.

[Lawrence et al., Fig. 1]

When the probe is a single gene, only that gene is expected to light up in FISH.  Figure 1 shows hybridization of a probe for the neu proto-oncogene in human lymphocytes. The  neu locus resides on chromosome 17. Since the cells contain diploid nuclei, two hybridizing spots are seen in each nucleus. The sensitivity of FISH is put into perspecive by considering the fact that each chromosome is a single DNA molecule. That is, each spot results from a few probe molecules hybridizing to different parts of a single DNA molecule. Looked at another way, we can recall that Avogadro's number (the number of molecules in a mole) is 6.02 x 10 23 molecules/mole,  we are detecting 1.66 x 10  -24 moles of chromosomal DNA!
 

Chromosome painting

[Cremer, Fig. 4 D,E]

It is possible to isolate DNA specifically from individual chromosomes. When total chromosomal DNA is used as a probe, it will base pair with all sequences on the chromosome from which it was derived, making the entire chromosome 'light up'. In other words, each sequence in the probe eventually encounters its complementary sequence on the chromosome and base-pairs with it.

Interphase chromosomes appear to occupy distinct 'territories'

See Chromosome Distribution within the nucleus from Learning Space at The Open University

http://www.open.edu/openlearn/science-maths-technology/science/biology/nucleic-acids-and-chromatin/content-section-8.3

 

The existence of chromosome territories reveals a fundamental level of organization in the interphase nucleus. It implies that as chromosomes decondense, rather than randomly unspooling their chromatin strands, a more orderly unpacking must occur, probably concurrent with the re-establishment of the nuclear matrix.
 

Discussion questions:

1)In this era of genomic maps and sequences, do we still need cytological banding and FISH methods?

2) Given that FISH technology exists, why bother learning about traditional staining methods?


 
previous page PLNT3140 Introductory Cytogenetics
Lecture 5, part 3 of 3
first page