(no subject)

[doyle]

It's all Greek to me, but I promised my connorbeast I'd ask for him:

Does anybody know what the substances in the BigDye® Terminator v3.1 Cycle Sequencing Kit and the substance Half BigDye do in gene sequencing? Either an explanation or a link to one would be much appreciated.

It's in relation to automated gene sequencing, apparently.

Comments

[circe-tigana.livejournal.com]

Connorbeast's smrt is so hawt.

[stars91.livejournal.com]

Not sure if this will help, but I found this link (http://www.appliedbiosystems.com/catalog/myab/StoreCatalog/products/CategoryDetails.jsp?hierarchyID=101&category3rd=112464&trail=no) at the applied biosystems website. Hope it helps.

[connorbeast.livejournal.com]

Thanks, that's the only website I could find too and it doesn't really explain it. Thanks again.

[julia-here.livejournal.com]

I was off to ask one of the S'cubies, who is a PhD doing research on fruit fly genetics, but I just realized she's en route to the North American Drosophila Association conference. How soon do you need the links?

Julia, hate it when my experts are unavailable

[connorbeast.livejournal.com]

Today sadly I need to have the thing finished for wednesday and this is one section holding me up. Thanks for trying.

[zandra-x.livejournal.com]

You might try at little_details. They know many unusual things.

Big Dye Sequencing

[(Anonymous)]

Hey, Julia sent me. I wasn't actually gone yet...not till Wednesday.

This may help - I'm gonna give a short review of how DNA sequencing works, too, so it'll all make sense.

Automated DNA sequencing works by taking advantage of the way DNA is replicated in living cells, and uses the same basic chemistry to add nucleotides (more generally known as "bases" - A, T, G, or C) to the end of a DNA strand. In a cell, you have double-stranded DNA, which is base-paired together (such that A pairs with T and G pairs with C, always). The cell separates the two strands, and then uses that fact to make a copy. That is, the DNA replication machinery sees an "A" on one strand, and inserts a "T" in the new one. If you copy both strands in this way, you now have two double-stranded molecules where you formerly had only one. (Example: If your DNA was "ATCG" paired on the other strand with "TACG", then the first would get copied to make "TACG", and the second to "ATGC", thus recapitulating both strands. The information required for the copying is inherent in the structure of the original molecule.)

So, DNA sequencing works much the same way. The same enzymes a cell would use are used to make a copy, but with a couple of tricks: first, you use fluorescently labeled bases, each of the four with a different dye, so that it's identifiable. Second, the labeled bases are also of a slightly abnormal, not found in living cells type, which can be added to a growing strand, but nothing can be added after them. Which results in "chain termination" when a labeled base is added.

So, you use mostly normal, unlabeled bases, but a small proportion of the labeled chain terminators. Then, every once in a while, you get incorporation of a labeled base, the strand copying stops, and you wind up with a mixture of strands of all possible lengths, because of random termination events at each position in the reaction. And since each base is individually identifiable, the machine that analyzes the data can say that strands of length 1 terminate with "T", strands of length 2 terminate in "A", strands of length 3 terminate in "C", strands of length 4 terminate in "G", and so on.

The Big Dye kits contain labeled dideoxynucleotides ("chain terminators") mixed with normal bases.

Hope this helps. Apologies for any jargon I didn't manage to weed out.

-Rachael

Re: Big Dye Sequencing

[connorbeast.livejournal.com]

Rachael I officially declare you the new love of my life. Thank you.

Re: Big Dye Sequencing

[(Anonymous)]

Hee. You're welcome. Glad it was some help. :D