3. Caryotyping by differential staining

The central task for cytogenetic analysis is to determine the karyotype. Karyotype - is the starting point of the study of the capability of all other methodological approaches. Study of chromosomes in the most general form can be carried out using various techniques routine staining, in which all chromosomes acquire the same color, evenly distributed along their length. This method allows to quantify violations of the karyotype, and identify some of the marker chromosomes, without specifying their origin. Because this approach is informative enough, the main method of karyotype analysis technique is G - banding of chromosomes (G - banding), which was developed in the late 1960s, Torbjorn Caspersson. The method relies on the acquisition of chromosomal pattern of light and dark bands along the length of the processing of trypsin followed by Giemsa stain color.

Striation character specific to each chromosome pair. The dark bands (G - band, G - bands) are formed at the site of interstitial heterochromatin. Such areas contain virtually no duplication of DNA and are rich in A-T pairs. In prometaphase, when chromosomes are at an early stage of condensation, we can count up to 2000 pages, in the second case - the bands 400-800 This method allows the identification of chromosome painting, identify deletions, inversions, insertions, translocations, fragile sites and more complex reconstruction. Staining G - pretreatment method prior cytogenetic preparations. Depending on the method it may be in the incubation buffer containing no Ca2 + and Mg2 + at t ° <= 37 ° C or t °> = 60 ° C incubation in solutions of proteolytic enzymes (trypsin, pepsin, etc.), incubation with deproteinionizing substances (urea, 2-mercaptoethanol), incubated in a standard saline solution (SSC) under the influence of alkali and heat.

There are different methods G - banding of chromosomes, but is commonly used GTG (pre-treatment with trypsin, stained with Giemsa stain). Exposure to trypsin provides transition regular helical structure in the irregular segmentation, which prevents perception Giemsa stain in G - negative areas and amplifies it in G - positive segments of chromosomes.

R - method provides cross striation chromosomes back to that in G - staining ( dark G - bands corresponding to light R - band and vice versa). Preparations obtained by R - staining, were analyzed using a phase-contrast microscope. Upon receipt of R - banding of chromosomes for pre-use Earle's balanced salt solution at t ° = 78 - 96 ° C, and certain pH values ​​and exposure time, which are different in different techniques. PREPARATION harder than G - banding, as often used preprocessing Ba2 at 60 ° C or NaOH and formaldehyde.

If the wire staining R - method at lower pH, and with the extension of the exposure, color only the telomeric region (T - method).

Q - differential staining of chromosomes obtained using fluorochromes that are being raised blue - violet light to mimic the red, yellow and orange glow. Of the dyes commonly used quinacrine, but better drugs are obtained by staining quinacrine - mustard or quinacrine - propylene slightly worse results are achieved with derivative bibenzimidazola Hoechst 33258. Strongly fluorescent chromosome regions corresponding to dark G - disks, the difference lies in the fact that the secondary constrictions of chromosomes 1 and 16 are colored only by G - method, and the intensity of the color segments of chromosomes 3, 4, 13 - 15, 21 - 22 and Y is weaker than when using Q - method.

If cytogenetic preparation put briefly in lye, and then for a long time to put in a solution of SSC at t ° = 60 - 65 ° C, will be painted only structural chromatin pericentromeric regions. This approach, known as C - the method used to detect pericentric inversions.