|prev page||PLNT3140 Introductory Cytogenetics
Lecture 18, part 3 of 3
Specific hybridization probes can be
made for each chromosome, allowing karotyping by "chromosome
When chromosomes are fluorescently-tagged, the amount of fluorescence is proportional to the size of the chromosome. Flow cytometers (Fluorescence-activated cell-sorters) 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.
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
can be separated from double-stranded DNA by HAP
chromatography. For some species, C0t-1
DNA is commercially available.
|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.|
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.
|Unless otherwise cited or referenced, all content on this page is licensed under the Creative Commons License Attribution Share-Alike 2.5 Canada|
|prev page||PLNT3140 Introductory
Lecture 18, part 3 of 3