Hi colleagues!
I need your help, because I have to understand the molecular combing method. I want to use the Molecular Combing system from Genomic Vision. Unfortunately, after reading several M&M sections I am still confused about several things; however I am happy for every answer to all or only parts of my questions. Thank you in advance!!!
1. How do I know in which orientation my single DNA molecule is placed on the silanized glass slide?
2. How much purified DNA material is needed as a minimum (or at least average)?
3. Is overlapping of single DNA molecules a problem?
4.I have problems to understand the results: I have labelled DNA molecules on my slide, ok. Next I hybridize with a fluorescent probe (Antibody or Probes), ok. But the readout is a long DNA fragment with several fluorescence spots. How do I identifiy a copy number variation? Do I have an internal reference?
1. You don't know, unless you use a series of probes that give an asymetric pattern.
2. I don't usually quantify DNA, but I used as few as 5000 cells in a 3 ml tank for combing, and it worked, but depending on what you do, it won't be enough (e.g. if you look for a specific, unique locus in the genome).
3. Again, it depends on what you mean to do. When studying DNA replication patterns, we work with low concentrations, to avoid overlaps, bundles, mixed patterns. If you search for a unique locus, you will probably have to increase the DNA density on the surface.
4. Apparently you are using a DNA barcoding kit. I don't know much about these, sorry. You should see missing bands for losses, and duplicated probes for gains. Check the location of your probes on the genome and compare to what you get.
Dear Vincent,
thank you very much for these clear and understandable answers!
Luckily it getting clear to me now, due to your help!
Thank you again!
I have one question left: Do you think it is possible tto identify a single DNA-molecule in a pool of hundreds of other single DNA-molecules? For example a single tumor cell in hundreds of health blood cells?
Thank you
1. I agree, you don't know the orientation unless you are hybridizing FISH probes that give a unique pattern that identify and orientate a specific locus.
2. for studies of replication dynamics we routinely use 100 000 - 200 000 cells per agarose plug.
3. overlapping of DNA molecules is a problem. Quality of combed molecules has to be checked by counterstaining of DNA with antibodies (also combing can be checked with Yoyo-1 staining). Bundles or overlapping of DNA molecules will give rise to aberrant signals (patterns) that mostly cannot and should not be interpreted.
4. Also I am not an expert of this.
Dear Hervé,
thank you also very much!
It helps me a lot to read your experiences and knowledge.
"I have one question left: Do you think it is possible tto identify a single DNA-molecule in a pool of hundreds of other single DNA-molecules? For example a single tumor cell in hundreds of health blood cells?"
>Again, I'm not an expert, but I know it's already quite a challenge to find one specific unique gene in a whole genome. If this genome is further diluted in a mix of different cell types, it gets crazy. You probably need to purify your tumor cells first, if possible :/
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques