there for many routine tasks in molecular biology laboratory today by many manufacturers kits. You have to isolate DNA? No problem, open your box, take a Some small plastic thing, then so much solution X on it by centrifugation, Y solution on it, done. That has to make over the old method of all yourself, of course advantages:
The results are generally reproducible, we have to handle no more dangerous chemicals such as phenol or chloroform, it is also faster.
The problem: Just because you no longer even works with chemicals, but only used ready-made solutions with meaningless names, many people do not know the application behind the kit chemistry. It will stop just proceed to rule. A second side effect of the kit is the lower yield. A good example to me is Next Generation Sequencing untergekommen:
In the new issue of Biotechniques, a paper by Maricic and Pääbo (login may be required) describes how a change to the standard 454 sequencing protocol can dramatically increase the size of the library of DNA that goes into the actual sequencing reaction.(Emphasis added.)
The trick used is to replace the last step in the library preparation where single stranded DNA is released from streptavidin beads. The original 454 protocol employes NaOH denaturation for this step, but the researchers found that this procedure results in a loss of over 99% of the DNA . However, When they replaced the NaOH denaturation with a heat denaturation by incubation recovery increased to 98%.
These authors are coming from the ancient DNA community and have an obvious motivation for optimizing the DNA retrieval from ancient biological material scarce. However, these findings are equally important to other applications Aimed at sequencing small volumes of biological material tested as tumors and within-host sub-populations of pathogens.
Wow. I'm used to that after DNA purification, only 50% rauszubekommen back, but> 99% loss? I'm speechless. 454 is one of Roche, and since none of the works will be paid to optimizing a process before it is released to customers? I wonder just how many people probably spent money on failed 454-sequencing have, and the reason was only one problem with the preparation of the DNA rather than the scientific question.
Maricic T, and Pääbo S (2009): Optimization of 454 sequencing library preparation from small amounts of DNA sequence permits determination of both DNA strands. BioTechniques 46 (1) :51-57.
(Actually I did last night, so post the paper review, then I have but a great recipe for peanut butter-chocolate brownies found which would test out I prefer.'s Why you get only used this short post.)
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