|previous page||PLNT3140 Introductory Cytogenetics
Lecture 5, part 3 of 3
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. Probes can either be synthetic oligonucleotides (eg. 18 nt) or larger sequences of DNA. The target sequences can be as small as 1kbp or larger than 15 kbp.
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 chromosomal 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 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!
[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 Universityhttp://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.
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?
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Lecture 5, part 3 of 3