SNP DNA sequecning

albertoruiz58 · Post by **albertoruiz58** » Fri Jul 11, 2014 1:57 pm

I am doing a science project that traces my ancestry all the way back to where our human species arose in Africa. I know how to extract, amplify, run it on a gel, and sequence it. In order to find my genetic markers (mutations) in my DNA where exactly do I need to look? Do i need to send it off to a lab to find the markers for me? am I able to do this myself? I need to find these markers in order to figure out what haplogroup I belong to. This is the most important part of my project. Thank You.

deleted-132180 · Post by **deleted-132180** » Sat Jul 12, 2014 1:56 pm

Hello there,

Sounds like a very cool project, but would require some sophisticated equipment for the procedures! If you are able to extract, amplify, and sequence your own mitochondrial DNA, are you actually working in a lab right now that has all the available resources to you? Because if that's the case, then the people in the lab would be the best people to ask as to how to analyze your data. What kind of resources are available to you, and where are you planning to work on this project because if you give us more details, we will be able to give you better advice as to what to do. I remember back in college, I did a similar exercise in one of my lab courses where we extracted our DNA, but afterwards just submitted it to a lab on campus which did the analysis of all the different markers for us and then told us what haplotype we are. They didn't give us much detail about what happened behind the scenes, so I don't clearly know how the analysis was done. Here is a bit more information that you can refer to (http://lslab.lscore.ucla.edu/MTDNA/), but if you have access to a lab space, I would ask the people around you what the best approach would be.

Let us know if you have anything else to add or if you have anymore questions.

Connie

albertoruiz58 · Post by **albertoruiz58** » Sat Jul 12, 2014 7:50 pm

This was very informative thank you! I have asked my Bitoech teacher after doing more research and it tunes out we do not have the DNA sequencing maching most labs use. I am able to extract my mtDNA, amplify it, and run it on a gel. I talked to my teacher and he said we are going to have to send it off to a lab. So, preforming electrophoresis is as far as I can take it in my lab at school so maybe you will be able to identify some polymorphisms. I will need to send the DNA sample off to The lab in order to be sequenced and told what my markers are in order to discover my haplo group. Can I send it off to any lab and they well tell me my markers and haplo group or is it just a specific lab? I have seen kits online but I am not sure. I kinda wanted to do it all myself but it should take less time off my project this way. Also, how would I be able to extract my y chromosomal DNA? I researched it online but I got confused and it seemed much more complicated than mtDNA.

deleted-132180 · Post by **deleted-132180** » Sat Jul 12, 2014 8:59 pm

Hello there,

I'm not quite sure which labs will specifically sequence mtDNA and tell you what markers you have. What you can do is do a little research of labs in local universities and see if any of them do work on mtDNA or knows a lot about sequencing, and you can contact them to see if they have any advice on what you can do. You can also contact various sequencing companies to see what type of sequencing services they offer and if those procedures are appropriate for what you want to do. As for extracting Y chromosomal DNA, I know that there are kits that allow you to isolate genomic DNA, but not sure if you can specifically pull out Y chromosomal DNA. Again, I would contact companies that sell DNA extraction kits to see if they know of any kits or technologies that exist that allow you to specifically pull out the Y chromosome. My guess is there probably isn't (but I could be wrong), and that you'd probably have to use primers to amplify different genes/regions in the Y chromosome and get those sequenced.

Hope that helped. Let us know if you have anymore questions!

Connie

albertoruiz58 · Post by **albertoruiz58** » Mon Jul 21, 2014 11:58 am

Thank you for your information you gave me about sequencing my mtDNA. I have found a lab in which I can send it off to one I have amplified after performing a PCR reaction. I am now on the final steps of establishing my procedure for my project. Once I have amplified my mtDNA I will need to perform electrophoresis. I have done this may times in school but have used only TAE buffer. In the DNA extraction kit I am using as a guide it says to use TBE buffer. I haven't heard of TBE buffer before. What is the difference and should I use it instead?

deleted-132180 · Post by **deleted-132180** » Mon Jul 21, 2014 12:16 pm

I personally have not used TBE buffer myself--I have always used TAE buffers for running DNA agarose gels, and my DNA extraction procedures have been fine. I did a quick search on the differences between TAE and TBE buffer, and here is something I found: http://www.researchgate.net/post/What_i ... rophoresis. I can't imagine using TAE instead of TBE would be a problem for DNA extractions, but just in case it is something specific to that extraction kit, I would contact the provider and ask them specifically whether TAE would be an issue for doing the extractions using their kit. Are there any experts here who have used TBE before and can offer some advice?

Hope that helped!

Connie

albertoruiz58 · Post by **albertoruiz58** » Mon Jul 21, 2014 4:09 pm

Thank you! That helped me out a bit. I figured there should be no real difference with using either or as well because I have ran many different types of DNA on a gel with TAE buffer and it hasn't been a problem. Thanks again.

albertoruiz58 · Post by **albertoruiz58** » Wed Sep 24, 2014 9:50 am

I am working on a project dealing with Mitochondrial DNA and focusing on SNPs within the DNA to determine haplogroups. I am perplexed on how exactly SNPs get their name/identity. For example, individuals in haplogroup C1b contain the SNP identified as A493G. How exactly is this mutation name? I know the A & G stand for the base pairs Adenine and Guanine but what exactly do the numbers represent?

deleted-226200 · Post by **deleted-226200** » Wed Sep 24, 2014 2:18 pm

Hi, Alberto.

The number represents the position/location on the mtDNA strand. So at position 493, there is an A to G base change.

You can find the standard mitochondrial sequence here, if you want to see the whole thing: https://www.mitomap.org/bin/view.pl/MIT ... manMitoSeq
The main site contains a lot of sequence and other information on human mtDNA.

Hope that helps!

-Lisa

albertoruiz58 · Post by **albertoruiz58** » Wed Sep 24, 2014 6:31 pm

That helps tons! Thank you for the website reference. That contains great information that I need. Thank you!!!

deleted-226200 · Post by **deleted-226200** » Tue Oct 07, 2014 4:21 pm

albertoruiz58 wrote:That helps tons! Thank you for the website reference. That contains great information that I need. Thank you!!!

Glad I could help. Good luck!

albertoruiz58 · Post by **albertoruiz58** » Wed Oct 29, 2014 6:31 pm

Hello. So, I have made great progress in my project. I have extracted and sequenced numerous samples of mtDNA from myself and and participants in my experiment. I have obtained the sequencing results and have begin to analyze the data. Through this analysis, I have discovered that one of participants has a c375t mutation. I know this because when I compared it to five other sequences, there was a C at this location and not a T. To me this symbolized a SNP and I though I could be able to find out information about this SNP. When I tried to research this mutation, I didn't find anything about it. I tried many different databases of information, including MIOTMAP. I was hoping that this mutation would help me discover this person's haplogroup and therefore their ancestry because it is a mutation. Am I doing something wrong? Is the science behind my project too complex for me to do what I want to do. I was hoping that for each SNP I found in each DNA sample that would lead me to finding there haplogroup, and I would be able to track their ancient ancestry with this information. I looks as though it's a lot more complicated than I perceived. I am lost at the moment and I would gladly appreciate your advice as to what I may be doing wrong, or what I should do in order for me to have my project succeed. Thank You.

deleted-226200 · Post by **deleted-226200** » Wed Oct 29, 2014 8:47 pm

Hi there. I didn't realize you were doing your own sequencing. That's fantastic!

I think this is the resource you'll want to use: http://www.mitomap.org/bin/view.pl/MITO ... smsControl
If you search for your mutation in the MITOMAP: mtDNA Control Region Sequence Variants, you'll see that there are 13 sequences in GenBank that have the 375 C to T mutation. From there you can click on the number (13) to see a list of the sequences which will also show you the predicted haplotype. You can also go to the articles that first published the sequences.

Hope that helps! Keep up the good work,

-Lisa

albertoruiz58 · Post by **albertoruiz58** » Thu Oct 30, 2014 9:33 am

THANK YOU!!!!!!!!!!!!! THIS HELPS ME SO MUCH!!!!!!! I am consumed with joy. I really appreciate it. This has done wonders for my project already. I really appreciate your feedback. Now my project is pure research from here, and I cannot wait to see all the information I will obtain. THANK YOU!!!!

deleted-226200 · Post by **deleted-226200** » Thu Oct 30, 2014 11:36 am

You are most welcome.

albertoruiz58 · Post by **albertoruiz58** » Fri Dec 05, 2014 9:49 am

Hello. So, I have completed all sequencing results of 25 individuals and have discovered their holographs. It took a lot of time and effort but it was quite amazing to see all the information I have obtained. Through bioservers.org, the place in which all sequencing results were listed, I was able to create a phylogenetic tree of some of my sequence results. The websites only allows 10 to be done at one time. I would like to make one big phylogenetic tree of all my results. Do you know of any online resources that could be used to make one with all my results? If not, I do have enough knowledge about my data and background knowledge of haplogroups so I think I would be able to construct one of my own but it would defiantly be time consuming. Also, I would like to ask your advice on how possibly I could take my project even further than discovering and individual's haplogroup and acient ancestry. I had many individuals that were in the same haplogroup, specifically haplogroup D, but made a discover that two individual's were in haplogroup D1j. I know that this means they share the same deep ancestral origins. I am stumped on maybe how to expand my project. I was thinking of showing something along the lines on how all the groups that diverged from macro haplogroup M and macro haplogroup N may be different and how they lead to the divergence of many different groups of peoples. I would love to have your advice. I have enough time before my project is due to maybe expand my project even more and actually discover something great as i feel that what I have done so far is great but is more of research. You advice has been wonderful so far, and I appreciate any help that you can. Thank you!

-Alberto

albertoruiz58 · Post by **albertoruiz58** » Mon Dec 08, 2014 5:18 pm

I am doing a project on the origins of my our human species and how we are all related as a species. What I have done thus far is that I have extracted, sequenced, and analyzed 25 individuals mitochondrial DNA. Through analyzing their DNA, I was looking for their Single Nucleotide Polymorphisms by comparing it to several of my other participants DNA sequences. When I found their SNP, I then searched it up on mitomap. A website which stores tons of information about the human mitochondrial DNA, including lists of haplogroups and their common SNPs. From here, I discovered each persons haplogroup and did more research as to what the origin of the group was, and what group it diverged from and when. I am also comparing which people are most closely related to each other based on the information I have obtain. I now obtain a huge chart of this data. What I need to know is, is how can I expand my experiment and make it innovative? My teacher just told me today that I need to make this more innovative because "even though what you are doing is cool and amaizing, it's not innovative." I have been researching and researching, but I really do not no more I can do. I have spent hours on what I've done so far and now I have until next Friday to obtain "actual data". So, if anyone could help me that would be amazing!!!!!!! What else can I do wit what I have done and obtained so far?

deleted-226200 · Post by **deleted-226200** » Mon Dec 08, 2014 8:22 pm

Alberto,

How exciting that you've generated data and you're able to get results from it. That is a great day in science!

If you do a google search on "phylogenetic tree makers" a bunch of results come up. You could look at the offerings and see if one of them would allow you to make a tree from more than 10 samples.

It seems you already have a pretty good idea on how you could expand your project. I think you should go for it. I have no specific suggestions for you.

Congratulations,
-Lisa

albertoruiz58 · Post by **albertoruiz58** » Mon Dec 08, 2014 8:37 pm

Thanks Lisa! I appreciate everything! I have obtained a huge chart of this data. My data chart lists each individuals SNP, its basepair on which it occurs, the haplogroup, when it diverged and from what group, and where it originated. What I need to know is, is how can I expand my experiment and make it innovative? My teacher just told me today that I need to make this more innovative because "even though what you are doing is cool and amazing, but it's not innovative." I have been researching and researching, but I really do not no more I can do. I have spent hours on what I've done so far and now I have until next Friday to obtain "actual data". So, if you help me that would be amazing!!!!!!! What else can I do with what I have done and obtained so far? Thank You.

deleted-226200 · Post by **deleted-226200** » Mon Dec 08, 2014 11:58 pm

Alberto,

You can check out the other phylogenetics projects on Science Buddies, especially the "Make it Your Own" tab, to see about things you could add on to innovate your project. Perhaps looking at some of the other projects will give you ideas.

https://www.sciencebuddies.org/science- ... ml#summary
https://www.sciencebuddies.org/science- ... eityourown
https://www.sciencebuddies.org/science- ... p017.shtml
https://www.sciencebuddies.org/science- ... p002.shtml
https://www.sciencebuddies.org/science- ... p012.shtml

Originally, it sounds like you wanted to do something on migration and population groups. Perhaps you could compare our data to the population in your area. Is it what you expected to find? Why or why not? And if not, what might be the cause?

Good luck,
-Lisa

albertoruiz58 · Post by **albertoruiz58** » Tue Dec 09, 2014 10:00 am

Okay. Thank you. I really appreciate your input!!! I will let you know how everything turns out.

sunmoonstars · Post by **sunmoonstars** » Tue Dec 09, 2014 11:09 am

Hi,

The work you did so far does sound very cool.

I'll try to give you a few ideas, but I am not exactly sure what your teacher is looking for.

Have you considered using the information to make predictions about the results from new samples?
Perhaps you can make predictions, then analyze how much (or how little) data you need in order to make an accurate prediction?

OR

Can you use the data to prove the samples were human?

I hope that helps.

Tonya

albertoruiz58 · Post by **albertoruiz58** » Tue Dec 09, 2014 4:46 pm

Hi, Well, I know all the samples were human because they were all individual human participants that all performed my experiment in which I led them through the DNA extraction. I thank you for the input I really appreciate it. I really do not know what my teacher want's me to do as well. He says this project really isn't innovate and it has all been done before. The thing is, I did this whole experiment by myself without and outside help and learned how to find SNPs, haplogroups, and discover everyone's ancient ancestry. I now have until next Friday to get data for December to make and my project "innovative". From my results, I discovered that I had a large amount of individuals who were in haplogroup D. So, I was thinking that maybe I could just focus on them but I'm just completely stumped. Maybe the mutations with this group code for different proteins, and I can somehow see what differences these show. I really don't know how actually do this though. Your ideas are helpful but I don't have time to make new samples. If you have more ideas please let me know.

deleted-71603 · Post by **deleted-71603** » Tue Dec 09, 2014 7:39 pm

Hello. We noticed you had three different threads going for the same topic. We merged them into a single thread. Although it seems you started a new thread with each phase of your experiment, it helps to keep all questions to one thread so that our experts can best help you based on what has already been discussed.

Good luck, and thanks for using Science Buddies!

albertoruiz58 · Post by **albertoruiz58** » Thu Dec 11, 2014 4:40 pm

I have completed an experiment where I extracted, amplified, and sequenced a few participants mitochondrial DNA. From here, I looked for each individuals SNP to discoverer what haplogroup they belonged to, to track their ancient ancestry. Now, I am trying ways to expand my project. I was thinking that sense I am dealing with SNPs maybe I can focus on how this changes the code for protein synthesis and different proteins are coded for or if the SNP stop the production of a protein. With this information, I can show how haplogroups can different from each other on the molecular level. I was told to use open reading frame software to help me out for what I want to do. I know how to use if but I am not sure where to go from there as I do not know the specific protein that is being coded for in the area in which a open frame is found. I am truly stuck. I would greatly appreciate any advice. Thanks.

SciB · Post by **SciB** » Thu Dec 11, 2014 8:33 pm

You said you amplified and sequenced mtDNA. Did you do a DNA BLAST to try and identify it? (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

I am not familiar with mt proteins but I'm sure SNPs in certain coding locations could alter the proteins and cause problems in mitochondrial function.

Check your sequences against the mtDNA database and let us know what you find. We may be able to help you identify the protein and determine if the SNP might alter it in some critical way.

Sounds interesting! Good luck.

Sybee

deleted-132180 · Post by **deleted-132180** » Sat Dec 13, 2014 6:19 pm

Hi there,

The previous expert already gave you some great advice. In addition, I would also look in the research literature to see if any of your mitochondrial proteins and SNPs of interest have been studied at the molecular level before (structurally, functionally, etc). That can also give you clues about how these SNPs may have altered the protein.

Let us know if you have anymore questions.

Connie

albertoruiz58 · Post by **albertoruiz58** » Sun Dec 14, 2014 5:53 pm

Thanks to the both of you! Always great advice! I will let you guys know ASAP what I find and if I have any further questions.

albertoruiz58 · Post by **albertoruiz58** » Tue Dec 16, 2014 7:53 am

So, I have used the blast web site using one of my DNA sequences. I had obtained 100 blast hits. The first hit I received was entitled this "Homo sapiens isolate Zapotec074 tRNA-Pro gene, partial sequence; D-loop and tRNA-Phe gene, complete sequence; and 12S ribosomal RNA gene, partial sequence; mitochondrial". I clicked om it and it showed me the alignment and it was off by one base pair. The when I clicked on the graphics it showed me that two tRNA sequences were highlighted. One was gttta tgtagcttac ctcctcaaag caatacactg aaaatgttta gacgggctca which coded for tRNA-Phe and the other was attctaattt aaactattct ctg which codes for tRNA pro. I am confused on where to go from here. What exteactly does this mean? Do the two sequences code for different things at these specific places on the DNA? Also there was a graphics tabs which I clicked on and it gave me this whole image which was kinda confusing to look at and I wasnt sure what i was looking at. I couldn't up load the image but here is the link: http://www.ncbi.nlm.nih.gov/nuccore/189 ... 9]&tracks=[key:sequence_track,name:Sequence,display_name:Sequence,id:STD1,category:Sequence,annots:Sequence,ShowLabel:true][key:gene_model_track,CDSProductFeats:false][key:alignment_track,name:other%20alignments,annots:NG%20Alignments|Refseq%20Alignments|Gnomon%20Alignments|Unnamed,shown:false]&v=0:1315&appname=ncbiblast&link_loc=fromSubj

So, not sure where to go from here and not sure what this all means. It is quite confusing to me. Any feedback would be great.

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