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Metaphase, interphase mapping
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Technique for chromosome gene mapping
Often, G-banded slides can be subsequently used for FISH (Fig. 9i), a technique particularly useful in mapping DNA probes on chromosomes. A few G-banded metaphases are photographed using a light microscope. Immersion oil is then removed by rinsing the slide in HemoDE, xylene or ethanol, for approximately 5-10 minutes. Slides are kept 15-30 minutes in PBS, 2xSSC or isotonic buffer at room temperature (rehydration) and then rinsed briefly in 70% and 100% ethanol (the alcohol will also remove the Giemsa stain). Afterwards, slides are pretreated with pepsin and used for FISH.
The main problems with this approach are the aging technique of the slide and the type of slide storage. Usually, G-banded slides are aged overnight at 65° C, then treated with trypsin and stained. The dry heat aging always impairs subsequent DNA hybridization. If either non-banded or G-banded slides are stored for years at room temperature (or in a dessicator), the material will become very "hard" and hybridazation will not be possible.
Chromosome position shift.
Chromosomes can shift and change shape and position during the denaturing process when subjected to the large temperature variations of the solutions. In Fig. 9i, the arrows point to a chromosome that clearly changes its shape and position after denaturing and hybridization.
Chromosome mapping of multiple DNA probes.
A simple procedure used for mapping multiple probes (cosmids, BACs) on chromosomes is triple color FISH. In this approach, probes are divided in "triplets" (groups of three) and one probe in each group is labeled with one of three different color (red, green and blue). Each triplet is hybridized at the same time on chromosomes, and the position of the probes relative to one another is recorded (see figure below). Later on, three, four or even more such triplets (depending on the color order showed by each triplet) are mixed together and hybridized at the same time. In the figure below, the drawings to the left show the principle of the procedure. In the middle, three "triplets" (image top) were hybridized together with two more probes (11 probes total) on the same chromosomes (image bottom). On the right side, 18 different cosmids were simultaneously hybridized and mapped using the same principle.
Interphase FISH mapping (on interphase nuclei)
When the physical distance between DNA probes is shorter than 500-1000 kb, their position relative to one another on metaphase chromosomes becomes more difficult to assess. To map DNA probes located less than 400-500 kb apart, one can use interphase FISH mapping. A convenient approach uses triple color FISH (see figure below). The probes are again grouped in triplets, and each probe labeled with a different color (red = R, green = G, blue = B). In the scheme at the top (step A), two triplets hybridize on nuclei. Results show that the red probe (R1) is in the "middle position in one of the triplets ( R1 is in the middle because in 50-100 nuclei, the distance between R1-G1 and R1-B1 is shorter than G1-B1. One can also measure that R1-G1 is shorter than R1-B1. In the other triplet, the green probe G2 is in the middle, and closer to B2 (G2-B2 is shorter than G2-R2). Because one does not know the relative position of the probes in triplet 1 from the probes in triplet 2, another hybridization is necessary to detect that relationship. In the second hybridization (step B), one probe from triplet 1 is mixed with probes from triplet 2 and vice-versa. After hybridization, in this hypothetical example, R2 is located between G1 and B1 but is closer to B1 than two G1 (in one hybridization) and B1 is located between G2 and R1, but closer to G2. If one combines this data together, the order depicted on the right side of the diagram is found. Such interphase hybridizations are depicted at the bottom of the figure. Because there are two chromosomes/nucleus, two such triplets can be scored in a nucleus. On the left, arrows depict the interphase hybridization pattern on two nuclei (nucl), showing the red probe between the green and the blue. On a rare chromosome (chr), we confirmed that pattern, and found that the blue probes was telomeric.In the example on the right, the chromosome shows a proximal green signal, with the position of the red and the blue probes impossible to differentiate. In a nucleus, one triplet shows blue in the middle, the other triplet shows red in the middle. This is the reason why one needs to count 50-100 such nuclei, to statistically determine which probes are closer to one another.
