Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
I was wondering something about genes and chromosomes as well. How can a gene be on different chromosomes? Are there different forms of the same gene that can be found on different chromosomes? Because I tried to search for the sequence of a particular gene in one of those gene databases and it turns out several options came up for that gene but they were on different chromosomes. Why is that? Then how do you know which gene is the one you are looking for?
Hmm... well some genes exist in multiple copies through-out the genome.
For example, human ribosomal genes are found in multiple copies of acrocentric chromosomes (in the sattelite region)... chromosomes: 13,14,15,21,22.
I can't think of any other genes that exist in multiple copies (at the momement) at different genomic locations (on different chromosomes).. but i'm sure that there are others.
hope this helps!
I see, but these genes don't exactly have the same sequence it seems? I'm reading a paper that talks about the interaction between these different genes, one binds to another, say gene A binds to gene B. If there are multiple copies of say gene A, how would I know which one gene B binds to? Or does it bind to all the copies on the different chromosomes? Thanks for the help!
okay... I'm just going to try to clarify your question (please correct me if my clarification is incorrect)...
you're saying... the paper that you're reading says that the protein A (encoded by gene A) interacts with protein B (encoded by gene B) ... and if there are multiple copies of gene A how would we know which Protien A protien B binds to.
..does this make sense?
Well, if you put it in terms of geneA/B and protein A/B i'm assuming that A and B are different genes (producing different protiens).
Sooo, i think you're asking if protein A is the result of several identical copies of gene A (at different chromosomal loci) how would we know the actual origin of the protein A in which protein B is binding to.
The simple answer is there is probably no way of telling which locus produces a particular copy protein A. Therefore, there is no way of telling which individual protein A molecule is bound by protein B (but of course ... there may be more to the story..ie. differential regulation at different genomic loci)
If you need help with whatever paper you're reading... post the reference (or PM me the referenece) and I can give you an "in-context" andmore precise explanation as to what exactly you're trying to decipher.
... anyways, i'll try ... to help you understand it. (but, I can't make 100% guarantees)
Right I was saying that Protein A and B binds to each other and since protein A is encoded by different genes on different chromosomes how would I know which particular gene on which chromosome to look at. There is a part two to this too- which I have posted on a separate post concerning something I read about PCR that you wrote elsewhere. You said the template cDNA we use are pre-extracted from cells and I was confused about what exactly this template DNA included. Would this DNA extracted from the cells include all the genes from these different chromosomes. If they were pre-extracted and sitting in the lab already does that mean they contain all the genes in our DNA and are sort of all purpose templates that can bind to many different primers or is the template only the specific cDNA sequence that binds to our primers we designed. I guess what I'm wondering is if the DNA was pre-extracted how would we know it contains the gene that we want amplified. Does that make sense? You're very kind Kim but I wouldn't want to put you through the torture of reading my papers, there are several and I'm more concerned about the basic principles behind this whole gene interaction thing rather than the specific genes they were talking about in the papers. I don't see how we would know which gene on which chromosome to choose to amplify that encodes for Protein A that binds to Protein B, unless it doesn't matter which one you pick because they would all encode for Protein A?
Hey there... i'm going to answer you question in parts... cuz you have a few of them
1.) "..... Protein A and B binds to each other and since protein A is encoded by different genes on different chromosomes how would I know which particular gene on which chromosome to look at. "
Well, the problem is there is no way of looking at which genomic locus a particular protein is derived from ... by just looking at the protien itself (assuming that protein product from all loci are identical)... HOWEVER, if you're interested in one particular locus... you may be able to design PCR primers that are directed upstream and downstream of that particular loci to selectively amplifyy it out of genomic DNA. (HOWEVER it is HIGHLY HIGHLY HIGHLY likely that all genomic loci have similar sequences in flanking regions [especially upstream] thus it may be difficult or IMPOSSIBLE to selectively amplify 1 locus while not-amplifying others ... you would have to know the gene sequences of all loci ... and then align the upstream/promoter region and downstream region to look for differences in order to design primers ... honestly,this just seems like a time consuming task!!!)
2.)" template cDNA we use are pre-extracted from cells and I was confused about what exactly this template DNA included."
First of all... I think Canalon replied to you on this one... (but, I suffer from short term memory loss so I can't remember what he said ... but I have faith that whatever he said in response to your question is 100% correct) So, pardon me if i'm being redundant....
Template DNA/RNA is any nucleic acid that you add to your tube too amplify during PCR. In the reaction... your 'template' is whatever DNA/RNA molecule that your primers anneal to... at the annealing temp. (this could be cDNA, genomic DNA, Plasmid DNA, mRNA etc.)
cDNA is something that is made in the laboratory. For example, I worked in a lab where we were interested in the transcription of fmr1 in response to smelling new things. To accomplish this I used 19 day old mice and exposed them to "mint-perfume" for different legnths of time. I then sacrificed them and removed their olfactory bulb. We knew that fmr1 levels changed in the olfactory bulb in resonse to learning new smells (because the olfatory bulb contains cells connected to the mouses' nose and brain). I then extracted nucleic acid and purfied mRNA from the sample. I then used the mRNA that I isolated from the mouse olfactory bulb and performed RT-PCR ... this protocol transforms the RNA into DNA ....and this particular type of DNA is called cDNA (thus the template for cDNA is mRNA)
3.) "Would this DNA extracted from the cells include all the genes from these different chromosomes. If they were pre-extracted and sitting in the lab already does that mean they contain all the genes in our DNA and are sort of all purpose templates that can bind to many different primers or is the template only the specific cDNA sequence that binds to our primers we designed. I guess what I'm wondering is if the DNA was pre-extracted how would we know it contains the gene that we want amplified."
Yes, if you're just lysing cells and extracting DNA you'll be extracting every ounce of genomic DNA from the cell. This includes all gene-DNA, exons, introns, flanking regions, all heterochromatic/non-coding/junk DNA from every single chromosome as well as mitochondrial and plasmid DNA contained in the cells that your'e lysing. If you were using pre-extracted genomic DNA ... it would be the same as if you lysed the cells and extracted genomic DNA yourself. Many times labortories will keep frozen stock of genomic DNA for PCR template (For instance.... if you were researching a type of plant that only germinated in january and was dead by february first... you would wait until mid january... collect TONS of this plant ... and then extract DNA ... save frozen stock in aliquots ... and use aliquotted DNA until next mid-january...)
Remember cDNA is based on the mRNA sequence, therefore .. primers will no anneal to flanking DNA regions or intronic DNA (because mRNA only containes exons!!).
Whereas primers designed to amplify from genomic DNA temlates can be designed to anneal to promoter (flanking) regions, introns as well as exons.
4.) "I don't see how we would know which gene on which chromosome to choose to amplify that encodes for Protein A that binds to Protein B, unless it doesn't matter which one you pick because they would all encode for Protein A?"
Well, if all genes encode for protein A ... I would like to say amplify any one ... however, I don't know what you're trying to do. So, i should say that I don't know enough about the context of what you're trying to do to give you my opion on that one.
5.) "... wouldn't want to put you through the torture of reading my papers, there are several and I'm more concerned about the basic principles behind this whole gene interaction thing rather than the specific genes they were talking about in the papers."
Tough papers only make you a better scientist ... and re-explaining them is an educational opportunity for both you and I. (yes ... I am a student too and DEFINITELY don't know everything!!!!!) So, if still want to pick out a paper for me to read i'd be happy to help you understand the parts that you're having difficulty with ... don't worry about it. (it wouldn't even take me 30 mins to "skim-through" a paper ... I do it all of the time when I'm trying to cram for an exam.. or picking out important points for a paper or talk)
Wow that was very detailed. Excuse me if I'm being stupid but basically that means if I designed primers based on a particular cDNA sequence I need to find out what type of cell the lab had pre-extracted the DNA from and make sure it contains the gene that I want? Basically, I am trying to look at genes and their expression levels that no one else had worked on before in the lab so I am not sure if the pre-extracted DNA that the lab has is appropriate for me to use as templates for my PCR reaction and whether they would contain the cDNA (or mRNA) that I designed my primers against. These genes are involved in focal adhesion and the we are thinking that when one is overexpressed the other should be as well since they are associated, so we are trying to check for that. So, I'm not exactly sure where the DNA should be extracted from. You said you were working on fmr1 involved in smelling so you extracted the DNA from olfactory bulb which makes sense. So if I were looking at genes involved in focal adhesion assembly/disassembly would I have to extract DNA from cells that form focal adhesions? The papers mentioned these genes being looked at in cancer cells, I think one of them from bladder or pancreatic cancer so should I be extracting them from cancer cell lines? Do both genes have to be extracted from the same cell lines or organs because I think these genes were looked at in differerent organs in different papers. Honestly, there really isn't a specific paper I was just looking at a couple to get an idea of what the genes do and how they were associated. I wasn't really looking into the experimental procedures there plus they don't really help me understand how to approach my specific question as to where I'm supposed to get my cDNA from, which seems more like a problem with my understanding of general experimental techniques involved. But you've been a great help, you have made many points much clearer to me.
Hey there... first of all sorry for taking SOOO long to get back to you. ( i ended up spending most of yesterday at the hospital ... I think i'll be fine after a little-bit-of surgery)
Anyways.. i'm back in lab a just started my first early morning PCR! AND i'm ready to talk about molecular bio. (my most favorite-est topic in the whole wide world)
.. i digress
Let me answer you questions in parts again
if I designed primers based on a particular cDNA sequence I need to find out what type of cell the lab had pre-extracted the DNA from and make sure it contains the gene that I want?
For there primer design you'd have to know which organisms to design your primers for. Primer sequences do not chang in a tissue specific/cell specific way within one organism. (Remember, each different tissue type contain ALL of the genes composing that organisms' genome. Therefore you'd use the same primers to amplify the same gene in mouse cDNA samples derived from both liver tissue and muscle tissus)
So if I were looking at genes involved in focal adhesion assembly/disassembly would I have to extract DNA from cells that form focal adhesions?
YES!!!!! you need to make sure that the cells (that the template cDNA is derived from ) produces/makes the types of focal adhesions that you're interested in investigating.
Do both genes have to be extracted from the same cell lines or organs because I think these genes were looked at in differerent organs in different papers.
Most genes are expressed in a tissue specific manner (gene expression in a eye cell is COMPLETELY different than gene expression in liver cells... and it's the patters of expression that makes a eye cell and liver cell different). Therefore it would be really difficult to make a conclusion/comparisons about 2 differnt types of adhesion molecules if they exist in different tissue types. (therfore you may get misleading results if your comparing mRNA expression in apples and oranges [mRNA expression in differnt cell/tissue types])
So, think of sticking with the same cell line, mouse type, cell type, tissue type when you design your experiment if your trying to compare the two tyypes of adhsion molecules. (ehem!!! stick with the EASIEST one possible!!!!! it makes life alot more bearable)...
well, i hope this was helpful.
Before i forget... I'd be really really really skeptical of making cDNA from from frozen stock/stored nucleic acid samples.
Maybe that's just my anal-retentive-ness.... but i'd rather work with freshly extracted nucleic acids. (using DEPC-added reagents whenever possible)
RNA and mRNA is EASILY degraded... so, any kind of improper care will result in RNA being 'eaten'
O my goodness, the hospital? Are you alright? I have also landed myself in the hospital a few months ago, if I didn't know any better I'd think school was making me sick. Anyhow, thank you responding to me eventhough you are only half-alive as I am. To give you an update, my primers were designed against human cDNA. I've decided to test a bunch of different cell lines derived from breast, lung, ovarian, and pancreatic tumors. I am converting the total RNA to get cDNA from RT PCR. You said RNA degrades easily if not handled carefully? I think I may have left my total RNA samples on ice to thaw for a long time, maybe about an hour would that affect the samples? I am worried that my RNA may have been "eaten up." I am also testing my primers, I've read all these suggestions about optimizing template and primer concentration and I am not sure why. If I checked my primers with normal PCR using the suggested amount of template (about 10ng?) and primer (I take 2ul from a 10uM stock) in a 100ul reaction and I see a band doesn't that mean my primers work? Why do I need to play around with the template and primer concentrations? Is that for the QPCR step? Do you have any suggestions as to what a good range to start with is? There are many different opinions on this but I would have more faith in your suggestions.
yeah, i think i'll be alright. It's just weird that something that seems like a "little" health-related problem has MORPHED. into a larger issue... but i'll feel better once everything is done (and my left upper eye-lid stops twitching!!!!)
Anyways, I'm glad you've got your project underway. So, do you know if these different cell types have the type of adhesion molecules? Are some of these tissue types going to be used as positive or negative controls? (ok.. i'm just being nosey.. you don't have to answer those questions)
So, i'm guessing that you're expecting to see a loss of expression of adhesion protein mRNA in the tumors. (when compared to matched normal tissue that should.. theoretically express more adhesion protein mRNA).
As far as degradation ... I personally don't think that I've left RNA on ice for over an hour. So, i'm not sure if your stuff has been eaten. However, if you have positive and negative controls... you can compare your experimental samples with your controls... and you might be able to deduce if your mRNA was in fact degraded.
You should optimize your concentrations so that PCR works efficiently ... Also, if everything is optimized it prevents smears of primers, primer-dimers, DNTP and non-specific (amplified) junk appearing on your gels. (This junk could also make future results difficult to interpret accurately)
I shoot for 10-15ng of template and to be honest with you I don't know what my exact primer concentration is (cuz I've never sat down and figured it out .... oi! i'm such of a slacker!!!!!)
But what I do with my primers is I look at how many nanomol I have (of lyophilzed primer) and I add 10 microliters of ddH2O per nanomole (so if the lyyphilized primer was 32nM i would add 320microliters of ddH20) and use this soultion as my stock-solution. To make my working solution I create a 1:10 dilution of my stock solution and then I add .5 microliters of my working solution for each 50 microliter of PCR reaction.
... anyways.. Idon't have my lab notebook in front of me.. and since I have a "bird-brain" my recollection may be off... (but i'll check tomorrow.. and repost if necessary)
BUT! the fact that you see a band suggests that your primers are working (just check the size of the band ... to make sure that the band represents the right DNA/PCR product).
Hey.. and if your conditions are working right now... don't change a DAMN thing!!!! The options exist as a way to optimize a reaction or make a reaction work 'cleaner' and trouble shoot the reaction (when it's not working at-all)... But if your stuff is working well now .. praise the PCR Gods and keep doing what you're doing.
good luck with everything!
Last edited by LilKim on Wed May 24, 2006 2:35 pm, edited 1 time in total.
Hi lil kim,
Thank you so much for your suggestions. I do see bands and the gel indicating my primers work, although there are some non-specific bands below it as well which I assume is just an excess of primer. I think now I am supposed to move on testing primer/template concentrations that will work best for QPCR. I am not quite sure what kind of curves I need to run, CT curves, standard curves, those are all terms that are circulating around my head but I think I have a bit more faith that this can be done =)
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