Molecular Cytogenetics - Science topic

1. Go to the UCSC Genome browser: https://genome.ucsc.edu/index.html

2. Mouse over "Tools" and select "Table Browser"

3. In the table browser window, set the following parameters: clade = mammal, genome = human, assembly = hg38 (or other), group = Mapping and Sequencing, track = Chromosome Band, table = cytoBand, position = chr1 (or whatever chromosome you are interested in), output format = all fields from selected table, file type returned = plain text

4. Click [get output]

The results will show the following 5 columns: chromosome number, cytoband start position, end position, cytoband name, and staining result.

Please check the following link.

I agree with Sonal opinion

Nick translation using commercial kits is in our hands as reliable as DOP-PCR labeling.

I am not sure what you mean by "bright signal region, which was blurred". Maybe you could upload a FISH-result?

Did you get a signal on one spot and it was just unsharp? If so maybe there is some problem with the chromosome preparation, or chromosomes are overdenatured.

20 Kb is more than enough to get a clear signal in case there is not too much repetitive DNA in your probe. (Many years ago the smallest probe I worked successful with was 0.5 kb of cDNA. In routine I used a 12 kb probe to detect reliably deletion or duplication in HNPP or CMT1a.)

K..S, Lavappa, a post-doc of mine, was in the process of clarifying the massive human cell contamination that had accumulated at the ATCC by the overwhelming HeLa cell contamination that occurred in a number of labs in the course of establishing neoplastic cell lines from human neoplasms.  Prior to his unfortunate death, he had amassed sufficient evidence following specific marker chromosomes of HeLa to demonstrate that all human neoplastic cell lines at the time were contaminants of HeLa. 

Lavappa's karyotyping was truly superb. While presenting his findings at the annual meeting of the Tissue Culture Association, the audience spontaneously burst to a lingering applause as he displayed truly superb Giemsa stained karyotypes displaying HeLa's marker chromosomes in all cell lines preserved at the ATCC. 

Earlier, Stanley Gartler caused bedlam at an earlier TCA meeting (Seattle?) when he clearly demonstrated, using the single and double banded features of the LDH enzyme that distinquishes the origin of cells derived from whites vs.blacks, respectively, by the single vs. double banded nature exhibited solely by HeLa cells.  All so-called human neoplasms cryopreserved at the ATCC had the characteristic double-band that is solely exhibited by tissues of black origin. Once Gartler stopped his presentation, all :"hell" broke loose in the conference room as individuals who derived the alternate cell lines rose up to defend their findings. It was truly a moment to remember. 

I suggest you look up K.S. Lavappa's publications to note if any of his technical steps may be helpful.

HeLa contamination was first reported by Ford and myself in 1958 at the time the Chinese hamster V79 cell line was derived. Gartler's publication is in Nature 217: 750, 1968.

George Yerganian (yerganian@cytogenhamsters.com)

Thank you Ilya. I will keep that in mind. Regards!!

Please look in to the attached protocol.

the best condition to spread the chromosomes is 50-53%  humidity and 28 C, according to our experience

luis

Dear Sirjan,

Sorry to answer so late but this answer could help others. Of course the quality of your extraction impact the quality of your labeling. In my experience the Life technologies kit is the best for high quantity and quality of BAC extraction. I use the maxiprep kit with small modifications (you have to increase the buffer volume of resupension, lysis and precipitation buffers twice regarding the maxiprep for plasmids and elute with the elution buffer pre-heated at 65°C). For labeling the Nick translation kit from abbott molecular, is the best (we've tried a kreatech technique that is awful! just as their probes!) and you have to perform the labeling during 16hours at 15°C. I can give the exact refernces of all products I use if someone's interested.

Good luck for your project

Best regards

It would be necessary to verify the chromosome countings on both populations. If the difference is proved true, you have can be two taxon different. It would then be interesting to verify the morphological characters in search of possible differences. We could explain this difference of chromosome numbers by phenomen as aneuploidy or dysploidy.

You can do it in old fashion way.  During the first mitotic division of embryo the maternal chromosomes and paternal ones  behave a bit differently and when you spread them you obtain two separated group of chromosomes. In this case they don't create  homogeneous metaphase plate.  Use air dried method of Tarkowski. Tarkowski A.K. 1966. An air-dryinf method for chromosome preparations from mouse eggs. Cytogenetics 5, 394-400. Their is a few modification of this method for different speciec.

Dear Dian,

without exact knowledge of your protocoll(s) it is not possible to make a proper troubleshoot! Most critical steps on your way to make chromosomes are:

1 Blood sample itself -> Heparinized blood NO EDTA-Blood! Direct after taking the blood the vacutainer should mixed head over tail a few times to avoid agglutination of the blood.

2 Culturing (72h-Culture) -> proper Conditions and Medium -> 1ml blood + 9ml culture medium (for example: RPMI 1640) + PhA. 1.5 hours prior to harvesting add Colcemid or Colchicin to the culture! 

If u want to establish synchronized "high resolution" culture you have to add MTX to the culture(s) at 12 to 16 hours before harvesting

3 Harvesting -> After spinning the cultures remove the supernatant, but leave about 1 cm of fluid above the pellet and do NOT remove the upper layer of the pellet coz here are the most metaphase cells!!!! Add about 8 ml prewarmed (37 degrees) hypotonic solution and mix by gently drawing the cells up and down with a Pasteur pipette and incubate for 10 minutes at 37 degrees or 15 minutes at room temperature (20 -22 degrees); mix gently once during incubation. After that spin and remove supernatant cautious, kleaving about 1 cm above the pellet. Resuspend cells carefully and start fixation -> hold vessel in a sloping position and start with 3 drops of freshly prepared ice chilled (-20 degrees) fixative (3:1 methanol:acetic acid) but DONT let them fall into the suspension!!! This step is very critical! Make sure that the first drops of fixative run down the wall of the vessel otherwise u might loose a lot of metaphases because they will burst if first fixation step is to rough! After u have added about 10 drops in that way mix gently with a Pasteur pipette and add another 5ml of fixative, mix gently and leave it for 10 minutes. Spin, remove the supernatant (red) but leav about 0.5 cm of it and add 8 ml fresh cold fixative, resuspend the pellet and spin again. Repeat this 1-2 times and after last soin remove supernatant except for the last 0.5 ml and resuspend cells.

4 Preparation of Slides -> important factors are: humidity, temperature and "condition" of ur slides. Start with 2-3 drops and air dry (or dry on dryplate) and check cell density under phase contrast microscope. If cell density is not sufficient just dilute or concentrate your suspension.

thanks Sebastian! I bought one named Synergy Taq DNA Pol from E-enzyme. I'm testing PCRs with that one to see how it work. 

Vasileios,

Tnah you for your helpful advice. Knowing that the slides will be good for the next few months till I process them is conforting. I chose to work with PK and so far so good. I do minor optimizations for different cell lines but in general it works without problems.

So far so good. Keeping my fingers crossed.

Jana

Hello Godfred!

I confirm Nallasivams hints. Furthermore you can optimize spreading in dropping procedure. Give cells time to soak completely with fixative. Moreover, a muggy atmosphere (by moist chamber at 37°C) in which the fixed cells should drop will help. Fixative is hydrophilic. In a moist atmosphere the fixed cell will soak with atmosphere water like a balloon. Thus, the chromosomes are separated more effectively in metaphases.

Good luck, Jörg.

Dear Mohamed, it depends on which kind of microscopy you apply. For convevtional semithin sections toluidine blue O is OK. For fluorescence microscopy, DAPI or propidium iodide are also very well.

Hello Sirjan!

Do you wash the slides in 90% and 100% ethanol after 70% ethanol?

This kind of reaction occurs when you put the slides directly into 100% ethanol after 2xSSC washing.

Regards,

Ilya

Humidity in cytogenetic FISH is crucial in two steps of the procedures- when you leave the slide overnight at 37 degree Celsius in dark chamber, and the other day when you remove the coverslip till placing the slide inty washing solution, the slide shouldn't dry

Dear Sulochana,

(1) No, you cannot replace dNTP mix for probe labelling with dNTP's from Fermentas Master mix. The problem is that in the Master mix there is a 1:1:1:1 ratio between nucleotides, and for labelling you need a mix with a lower concentration of dTTP, which will allow to incorporate fluorochrome dUTP into the amplified DNA product.

(2) No, it is impossible to replace flourochrome-dUPT with flourochrome only. Such fluorochrome (for example Alexa) woud stain DNA but not label the DNA.

(3) 500-1000 bp long means the size of used DNA fragments.

Hope it helps.

Best regards,

Frantisek

Hi,

Is this copy number neutral LOH or is it LOH associated with a loss Xq material?

In any case, in combination with skewed inactivation you can have a good lead to find mutations.

Are there similar cases in Decipher?

kind regards,

Karoly