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Lecture 18, part 3 of 3
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D. Chromosome Painting

Schröck, E. et al. (1996) Multicolor spectral karyotyping of human chromosomes. Science 273: 494-497.

Specific hybridization probes can be made for each chromosome, allowing karotyping by "chromosome painting".
 

1. Chromosome-specific DNA

When chromosomes are fluorescently-tagged, the amount of fluorescence is proportional to the size of the chromosome. Fluorescence-activated cell-sorters (FACS) illuminate chromosomes with a laser, and the amount of fluorescence is detected. Depending on the intensity, a chromosome leaving the sorter is deflected into a particular tube. The result is that all 24 human chromosomes can be purified. By PCR with random primers, the entire DNA complement of a given chromosome can be amplified, to produce small samples of DNA from a single chromosome.

2. Suppressor blocking probe.

Since most of the human genome is repititive DNA, total chromosomal DNA from any one chromosome would be expected to hybridize with all chromosomes. On the other hand, low copy number sequences from each chromosome should be unique to that chromosome. To saturate repetitive chromosomal sequences during Fluorescence In-situ hybridization (FISH), chromosomes can be hybridized with non-labeled probe from repetitive DNA, leaving only low-copy number sequences unhybridized. The low copy sequences are free to hybridize specifically with low-copy sequences from the probe.

Blocking probe is made by allowing unlabeled total human DNA to anneal to C0 t = 1, at which low-copy DNA will remain single-stranded, but repetitive DNA will be in duplex form. Single-stranded DNA can be separated from double-stranded DNA by HAP chromatography. For some species, C0t-1 DNA is commercially available.
 

3. Labeling with dye combinations


Because there are 24 human chromosomes (counting X & Y), and only a few dyes available for fluorescent labeling, each chromosome must be labeled with a unique combination of dyes so that a distinct emission spectrum will be obtained for each chromosome. Typically 5 dyes are used: Cy2, Spectrum Green, Cy3, Texas Red and Cy5. For 5 dyes, there are 25 = 32 possible combinations. Thus, a probe made with only 1 dye might have a single peak emission wavelength, while a probe made with 3 dyes might have peaks at 3 distinct wavelengths. By measuring the emission spectra at each pixel in the image of the chromosomes, visualized in fluorescence microscopy, a computer program can determine which chromosome-specific probe produced that pixel.
 

4. Hybrization



from http://wsrv.clas.virginia.edu/~rjh9u/colrkar.html by Robert J. Huskey.

FISH is performed using a mixture of the 24 chromosome-specific probes, and a large excess of blocking probe. Although the labeled chromosome-specific probes also contain repetitive sequences, the repetitive sequences on the slide are saturated by unlabeled probe, allowing very little of the labeled repetitive sequences to hybridize. Consequently, only low-copy number labeled sequences will be unblocked on the slide, and only chromosome specific sequences will hybridize.

The chromosomal image is acquired by a CCD camera, and a computer program determines the emission spectrum for each pixel. Based on the spectrum, that pixel is assigned a color for classification purposes,  resulting in an image that shows each chromosome to be a distinct color.

As we will see in Lecture 19, chromosome painting makes it easy to detect chromosomal abnormalities, such as translocations, deletions, or inversions.

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prev  page PLNT3140 Introductory Cytogenetics
Lecture 18, part 3 of 3
first page