About getting a genetic sequence

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bbs_r
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About getting a genetic sequence

Post by bbs_r » Mon Aug 08, 2005 2:53 pm

I'm an undergraduate. I apologise that I maybe asking a stupid question, but I'm curious actually.

Assume I got an unknown organism and I would like to have its genetic code, can anyone tell me what to do next after getting its DNA, which acquired after centrifugation?

Thanks a lot! :)

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Post by sdekivit » Mon Aug 08, 2005 5:11 pm

you can, for example, apply the dideoxy-method of Sanger and separate it with gel electroforesis. But another method that is used is fluorescent labeling. But i think for you the Sanger-method is the most interesting.

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Post by mothorc » Wed Aug 10, 2005 5:38 am

did you you have the RNA ssu DNA?
I think you must think more about your study. I mean you must find and read a lot of articles before start if you don't want to be a blind. A best plan is better. It is my experience.
Good luck to you.
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Post by deedeesofia » Thu Aug 11, 2005 4:19 pm

your question is kind of...hazy. so, you want to sequence the dna.
how big is the genome of your organism? do you want to sequence the entire thing the way they did with the mouse, human etc? or do you just wnat to sequence a couple of kb?

In a lab, you can routinely squence...700 bp at a time/per reaction, so it depends how much DNA you want to sequence!
if you wnat to know more, let me know! we sequence regularly, but like I said, not entire genomes!!

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Post by MrMistery » Thu Aug 11, 2005 6:11 pm

When i was at the national lot i went to the national research centre. There they had a machine called "DNA sequencer"(guess what it does. Hint:it doesn't make espresso :D ). An incredible thing: you just put the DNA molecule there and it gives you the whole genome(i assume it has limits though..). At a measly 50.000 euros...
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Post by chemistry_freako » Fri Aug 12, 2005 3:48 am

hmms but Sanger's dideoxy method of sequencing does seem like an option u could use - it basically involves the use od dideoxynucleotides (ddNTPs) in addition to the normal nucleotides (dNTPs) found in DNA. Since ddNTPs don't have a 3'OH end, but instead have a 3'H end, further addition of nucleotides to the strand cannot carry on.

http://www.bio.davidson.edu/courses/Bio111/seq.html
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Post by canalon » Fri Aug 12, 2005 3:59 am

Automatic sequencing uses the dideoxy method... But they just use a machine that can make the reactions, run capillary electrophoresis and read the results without human intervention. The classical sequencing by hand and with radioactively labeled terminating ddNTP is a pain with 4 runs (one per ddNTP added in the mix), with a machine, it's faster and cleaner (do not use radioactivity but fluorescent labeled ddNTP, with four colors, so that you can read the sequence in one run, and one reaction, not 4), but you still need clean DNA (And I am having trouble those days having that....) and a good tech (I won't comment on that either...).

But even with the cleanest DNA and a good technician you will have trouble reading more than 1500 base stretch, far from a complete genome. For that you need plenty of time and a usually a lot of machines (You should search about TIGR: the institute for Genomic Research). Not as easy as you seem to believe Andrew.

Cheers

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Post by MrMistery » Fri Aug 12, 2005 8:24 pm

Sorry sir, my bad... :lol:
I don't know that much about experimental genetic yet, Patrick. Actually i don't know anything. But i would like to learn someday. Anyway it will take many years until i will know as much as you since this is what you do to put food on the table.
Well, that's it from the noob....
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Post by DevGrp » Fri Aug 26, 2005 9:18 am

You now have your organisms DNA, a whole bank of Applied Biosystems 3730xl 96-capillary machines and shed loads of money. The only thing you need to sequence now is some known sequence to start you off. Sequencing requires a ploymerase to copy your DNA and DNA ploymerases need somewhere to start from. In sequencing (and PCR) this usually takes the form of a short oligonucleotide of known sequence (primer) which binds to your target DNA and starts of the new stand.

The simplest way to get round the need for known sequeunce to start off the reaction is to chop your DNA into chunks (using resitriction enzymes) and then clone these chunks into a vector of some sort. Plasmids are the simplest but you can also use baceteriophage, or artificial chromosomes (BACs and YACs). Now as you know the sequence of your vector you can design primers based on this and start your sequencing off. (infact there are a whole series of predesigned sequencing primers called things like M13 forward, M13 reverse, Universal forward and reverse, T7, T3 etc which you can buy of the shelf.). If your DNA is in a circular plasmid you can start sequencing it from both ends. Once you have got your sequecning results you can then use your new sequences to design new primers and sequencing even further into the DNA inserted into your vector. This is called primer walking.

In our lab we used to do sequencing ourselves (first with manual plates and radioactive labelled ddNTPs and then with smaller automated machines but to be honest, these days, unless you are a specialist sequecing lab (and even our insitutes Technology facility couldn't make it pay) it isn't worth the trouble and we just send off our samples to a company that specialises in sequencing and get our results back in a couple of days at the cost of £10 a sequence.

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Re: About getting a genetic sequence

Post by CXCR4 » Tue Aug 11, 2009 9:36 pm

I am no expert but after genetic sequencing I would try to compare it's DNA to our human genome first. Before I did any thing else that way you could understand the organism vs. human beings. It's funny because I have an experiment as well with a totally different species but I don't know how to completely extract the DNA. But I do know about the Centrifuge with blood plasma Separation. I say this because the subject is more of a symbiotic species. I mean the species acts as if it is more of a host therefore it could have a resistance to human diseases as well as being venerable to diseases it self. How long have you been a genetic analysis? If you don’t mind what class is the organism placed in, or what class do you think it is placed. I am working on the same thing but dealing with work and school I fail to make time for myself. Are you full time in this field of study, I like the thought process of neu species. I have many myself, but have lost many of them due to a personal Lab, lol ha

Oh by the way what is your Online Name?

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Post by jyaron » Wed Aug 12, 2009 12:46 am

As others have said it isn't exactly easy to sequence a genome.. DNA is only slightly easier. If you are trying to classify your organism it is best to try and go the cheaper and easier (albeit more ambiguous and longer) way and try to identify specific genes within your sequence's DNA. For example, try to compare it to a human housekeeping gene such as GapDH or HSP70. If it doesn't contain it, then it is most likely not a human cell.

And so on.
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