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Can you use mtDNA to...

Genetics as it applies to evolution, molecular biology, and medical aspects.

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Can you use mtDNA to...

Postby Swordy » Wed Apr 22, 2009 5:42 pm

Is it possible to use mtDNA to determine if two different individuals of the same species are related and how closely related they are?

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Postby MrMistery » Wed Apr 22, 2009 11:48 pm

generally, you can. But you can only do this by comparing them with other individuals. If you just know the sequence of their mtDNA, that alone won't provide a lot of info, unless the molecular clock for the mtDNA genes of the particular species has been described.
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Re: Can you use mtDNA to...

Postby Swordy » Thu Apr 23, 2009 8:49 pm

What do you mean Molecular Clock?
And can you give me more specifics on how this works? You just extract the DNA, PCR it, and Sequence it?
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Postby MrMistery » Fri Apr 24, 2009 3:17 am

molecular clock - each gene has a ~constant rate of mutation.
and I don't know about the exact protocol, I don't do this kind of stuff. but i think that basically that's how things work.
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Postby Swordy » Fri Apr 24, 2009 8:15 pm

Well i have done some research on this a little bit and it lookes like this is only useful in determaning if 2 animals are of the same Species... im more needing how closely related 2 animals of the same species are, like if they share the same mother or if they are completely unrelated... seeing as how both you and i are not experts or even know much about this field does anyone know more on this cause it would be very usefull to know?

*edit*
http://damian.peterson.net.nz/2009/04/0 ... -nutshell/

I found that when digging alot deeper... does this seem right?
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Postby MrMistery » Sat Apr 25, 2009 4:03 am

i just did mtDNA typing last week and found out that i am more related to germans than to Yoruba africans (duh, I'm european). so it is possible to do it for two organisms in the same species.

Mitochondria are generally useful for the same species, because they have a very high mutation rate.
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Postby wbla3335 » Sat Apr 25, 2009 8:06 am

Hi Swordy,

To address your initial question, mtDNA can be used to determine if two individuals are related - within limits. And it can be used to determine how closely related they are - within limits. Barring new mutations, mtDNA within any species will exist in a limited number of different haplotypes (mtDNA is haploid). That is, there are a limited number of different sequences of the mtDNAs within a species. All individuals differ in the sequences of their nuclear genomes due to recombination during meiosis and the union of sperm and egg. But mtDNA doesn't undergo recombinatiion. It is passed from one generation to the next intact (barring new mutations). It is also inherited maternally. The sequence of your mtDNA is the same as your mother's, so you can determine if you are (likely to be) the child of your mother by sequencing mtDNA. Your father, though, is likely to have a different sequence. Let's say that a species has a million individuals. Within this species there may be a thousand different mtDNA haplotypes (I have no idea what an actual proportion might be). So if two individuals have the same haplotype, they are likely to be related. If one mtDNA has a haplotype that differs from another by a single nucleotide, then these are likely to be more closely related to each other than to an mtDNA that differs by five nucleotides. Building a genetic family tree from mtDNA sequence data, though, is difficult. What you will get is a tree of haplotypes. Two individuals that have the same mtDNA haplotype, though, could be more distantly related than two indivduals that have different haplotypes. Having the same mtDNA haplotype just means that you belong to the same maternal lineage. Your cousin on your mother's side of the family may have the same mtDNA that you have, and your cousin on your father's side may have a different mtDNA, but you are equally related to those two cousins. Genetic family trees can be built, but you will have to get information from the nuclear genome, and lots of it.
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Postby Swordy » Mon Apr 27, 2009 5:41 pm

hmmm so you sequence the mtDNA and based on how similar it is you can tell how closely 2 organisms are related within limits? Interesting.... what are the limits if i may ask? Also i asked my Biology teacher about this and he told me something about "The HyperVariable Region" and it might be useful here.... he was quite vague and not really sure So do you know anything about that?

Also what Nuclear information would i need to get and Please be specific. :)
Also what part of the mtDNA Should i try sequencing or should i just do it all?
I Can use all the information i can get on this subject so anything at all might be useful :)
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Postby wbla3335 » Tue Apr 28, 2009 12:45 pm

The limits of using mtDNA for determining who is more closely related to whom is that there are a limited number of haplotypes present in a population compared to the total number of indivduals in the population. Two individuals might have the same mtDNA sequence, which means that they are part of the same maternal lineage, yet one may be more closely related to a third individual. As a simple example, consider a child, its mother, and its grandmother. All three will have the same mtDNA because mitochondria are maternally inherited, yet the child is more closely related to its father, who may have a different mtDNA, than to its grandmother. mtDNA is generally good for comparing populations within a species, and can be used for phylogenetic analysis of more distantly related groups such as species, genera, and families. Due to its high rate of mutation, though, there comes a point where mutations begin to occur at sites that have already mutated, and the stories they tell begin to get a bit jumbled.

Your teacher was referring to the control region, also known as the D-loop, of mtDNA. This region does not contain any genes and so is the most variable region in the mitochondrial genome. It accumulates mutations faster than other regions and so is best for comparing closely related organisms.

The best nuclear DNA to use for determining relationships between closely related indivduals is, again, the most variable regions. Introns, pseudogenes, MHC genes would be your best bets. May I ask what species you're working with? And what your goal is? Microsatellites might be better suited to your purpose than sequencing DNA.
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Postby Swordy » Tue Apr 28, 2009 5:32 pm

They are Chiricahua Leopard Frog (Rana chiricahuensis) eggs. Microsatellites have been tryed on the eggs but due to the Unfirtilization of the eggs there is only one set of Cromosomes to try to PRC making it so i can not transer from one tube to another.... I currently have my Single Tube Method up in the Molecular Biology Forum asking for suggestion if you wanted to read up on that...

uh my goal is to be able to determane if two frog egg masses are related due to the fact that all the frog eggs AZ Fish and Game has sent me are horribly labeled and it would save alot of work later to know which frogs have the same parents if you are able to just test a few before there starting to grow so some organization can be used to reduce the number of tests needed on every single fully grown frog.......

That answer everything you need?
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Postby wbla3335 » Wed Apr 29, 2009 12:54 pm

Well, if there isn't a microsatellite system up and running for your species, it wouldn't be worth your time to develop one. I assume that the egg masses are fertilized and that each mass is fertilized by a single male (if the mating behaviour of your frogs is similar to the norm for frogs). And you can only get a single PCR reaction from each egg. (I had a look at your single-tube protocol. Have you tried this yet?). Maybe you could type the mtDNA (sequencing, RFLP, SSCP) to get maternal information, and find a variable region of the male sex chromosome (that has no corresponding region on the female sex chromosome - is this possible in your species?) to get paternal information. Whether this would be enough, I don't know. You may need more nuclear information to be able to identify individual fathers. I know that many amphibians are polyploid. Do you know the ploidy level of your species? Are these egg masses from one pond/lake, drainage sytem, state? What is important is if you need to discriminate between individuals of a single population or many populations. It doesn't sound like you need an actual tree that may indicate the degree of relateness of all the egg masses, but just whether one differs from another.
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Postby Swordy » Wed Apr 29, 2009 5:33 pm

If by MicroSatellite System you mean a DNA primers that copy microSatellites then we already have that and we had a poster of our Findings at PAG this year....
I honestly think the eggs they gave me are not firtilized or maybe they tried to firtilize them and it didnt work or something... or atleast thats the leading theory we have in the group....
uh yes you are correct in a way and wrong in another... Our Group found a way of making a triple reaction that puts 3 primers in a single tube and to copy 3 MicroSats at the same time, but once we have that there is no other uses for the Extract cause all of it was exposed to the Primers, Taq and other PCR ingredients and is pretty much useless after that....
I have tried my Single tube protocol many times on the eggs with no success, however all those times i was going after MicroSats in the eggs, and i have a new extract in the Freezer right now that was targeting to see if there was mtDNA in the Eggs... our lab is out of Agarose right now so i cant run it and some other samples out on a gel to see if it worked. However if it didn't work in any way im going to try it agian with out the SDS in case that messed with the reaction, and if that doesnt work then im going to say that they gave me messed up eggs...

uh the other info you wanted is going to take me a bit to find out as i honestly have no idea what half that means so im going to go ask my Bioteacher/ Project leader about that and i should have that in a few hours....
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