CharonY

I just hope that that was not serious.

DNA regions encoding a protein do not, per definitionem, contain regions upstream of the coding sequence...

contaminations

In part,yes. Of course for eukaryotes you'll use a poly-T primer, in that case the primer bias is lower. Yet, efficiency of RT is also dependent on the template itself. Secondary structures can be a problem, for example. Also your RNA may degrade during your RT reaction, so that certain instable low abundant RNAs are getting lost, introducing yet another bias. Despite all that for certain techniques (e.g. microarrays as well qPCR) you have to make an RT step, though. It is only important to keep in mind that a bias is possible (and even likely). Regarding housekeeping genes: afaik there are hardly genes that fit the bill. This is especially true for stress experiments.

A former colleague of mine is actually doing PCR on beads (for 454). The PCR is done in an emulsion. Maybe this paper helps. PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets Takaaki Kojima, Yoshiaki Takei, Miharu Ohtsuka, Yasuaki Kawarasaki, Tsuneo Yamane and Hideo Nakano* Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Nucleic Acids Research 2005 33(17):e150; doi:10.1093/nar/gni143

Good point. Actually funny how the focus of one's own work influences the interpretation, though (I am more of an RNA/Protein guy...).

Depending on what he wants to compare this is not an option, as RT can induce a bias in the level of detection (so basically it is a matter of normalization options). It might be easier if a protocol is posted.

I think he is referring to effects of the sequence on, say splicing, mRNA transport or translation. One example that I know of and which is quite intuitively understandable is for instance a silent mutation changes to a rarely used codon (but leading to the same AA). So if the given tRNA is less abundant in the cell, the translation is slowed down apparently leading to a different, less efficient conformation. This has been demonstrated for the MDR1 gene, for instance. (Kimchi-Safarty, Science 2006).

Upon rereading I found that I misunderstood you. I assume I was thinking of ESTs for some reasons (or for any other reasons I assumed you meant to sequence exons or whatever). Of course RACE or other essays essentially only lead to same that you propose: getting the sequence of the mRNA. My apologies for the distraction.

Well, it depends. If I refer to mRNA I usually mean the complete transcribed molecule. That is, including the upstream non-coding regions (including promotors and RBS). But this might only be a matter of terminology.

But mRNAs do not start with the exons, wouldn't a RACE PCR for instance be more viable for #3?

You add proteases to a) clean up your DNA extract from some proteins in general and b) DNA binding proteins in particular. DNA is wound tightly around histones and if this association is not disrupted, you will find protein contamination in your DNA sample. What is the question for the next two parts?

The parents are not homozygous.

[nitpick] You mean mammalian erythrocytes, right? [/nitpick]

nope, I copied it from another site, but then I probably just overlooked your quote. You will note however that the definition of the NAS is not precisely the same as Futuya's, which you brought forth in the first place. And these two are not the only definitions that are currently used by biologists. Well, I thought I implied it. Here essentially Futuyma states that biological evolution begins with changes in allele frequencies and ends with the diversity of species as we have now. This reflects my point of view also. Apparently this is the main point of our disagreement, though. Yet if one explores the statement "descent with modification" closer I'd say it is not too far from change of allele frequencies over time. It does not (at least as I would interpret it) require speciation for instance. And everything below that would be a shuffling of alleles. Yet, it does not define how much shuffling is needed to accept it as a evolutionary process. And this in turn also has an impact on evolution of proakaryotes. Here you do not have easily definable endpoint frequencies (e.g. fixation) due to extensive horizontal gene transfer. BTW the genetic species concept as applied to prokaryotes is an extremely arbitrary distinction (70% hybridization). re fixation: Well I am apparently misinterpreting you but this: I'd rather say that it is one accepted definition of evolution. As you will note from lucaspa's posts it is not undisputed (and rightly so). I'd say that changes in allele frequencies is the minimal definition because this is the proximal result of evolutionary effects. On the "maximum" side one could argue that once we obsever specietion we know that evolution happened.

Actually I do not think that any poster argues differently (even the non-American ones). Yet, it should also be allowed to criticize what they say (especially if one is as incoherent and illogical as Coulter). On another note, I assume people do not like to work at Walmart due to the bad working conditions.

In this very interesting paper it is indicated that at least in Pseudomonas aeruginosa anitbiotic sensitivity accumlates faster than resistance. This indicates that there might not be a trend for accumlation of resistances. Quite the contrary as usually expected! Antimicrobial Agents and Chemotherapy, July 2006, p. 2506-2515, Vol. 50, No. 7

Well Futuyma's concise quote regarding evolution is: I copied that from somewhere else because frankly, I cannot find my copy atm. This quote (especially the last sentence) implies that biological evolution happens within a continuum including also simple alterations of allele frequency (with fixation being the endpoint of such a process). One has to keep in mind that "changes in allele frequency" is not the definition of evolution per se, but it can serve as a minimal definition. Regarding species while what you said has some truth in it, you have consider that there are inherent problems with the definition proposed by Mayr. Take prokaryotes for instance. I assure you, you will have problems defining a species concept that suits them. Moreover, evolution and species concepts are to some point intertwined. You need somewhat closed populations in order to observe evolution and in turn evolution is the reason why different populations exist in the first place. So a fuzziness regarding species will also have an impact on the definition of evolution. I'd be glad if you can prove me wrong but so far I did not ran about anything like sharp, universal definitions for both concepts... Actually how did we come to this. Oh yeah, wheter evolution is only happening when fixation occurs. right.

In theory you are correct, dak (correct me if I am wrong, Bluenoise). In case of known sequences (for screening purposes) one could indeed try complete digests with a number of enzymes combinations, but with a sequence of a couple megabases it can get a bit tricky. Using singular enzyme the fragments will probably too short for compelte digests (~256 bp for a tetrameric recognition site). If however, the library is intended for sequencing purposes there is hardly another way than incomplete (hopefully) random digests.

Suppose for that small fragments it is the way to go. First a couple of test runs to estimate digestion rates, then resolve amd and elute appropriate band(s) from gel.

Maybe putting it a bit simpler, organisms that do meiosis have (at least) a diploid genome, meaning they posses two sets of a given chromosome (one from each parent). During meiosis the pair is split and the offspring will only get one pair (and the other one from the other parent). Prokaryotes only posses on chromosome which is replicated and given to the daughter cell asexually. Therefore there is no need (or possibility) to split chromosome pairs.

Well with DNase I (as with every other partial digestion for that matter) you have to carefully time your digests. More importantly, you have to redo it for every new batch of enzymes (within a manufacturer often but not always it is reproducible, though). Regardung DNase I specifity: as I mentioned a bit obscurely the interaction of DNase I is dependent on the local structure of your DNA strand and less on the sequence. More precisely it is sensitive to the structure to the minor grove. There is (at least afaik no interaction with the bases per se, which is the reason why DNase I does not recognizes sequences. So it does not simply find a pyrimidine and cleaves there, which would, as you pointed out, yield theoretical cleavages at every site. Instead the rigidity and depth of the minor grove are the parameters that are recognized by DNaseI. But as you are aware of, these parameters are of course sequence dependent. More importantly though, the groves are unsymmetrical structers as such the position of, in this case, a pyrimidine on one strand is not equal to the presence on the other one. Thus if you have got certain number of pyrimidines (or maybe it was pyrimidine-purine transitions, I forgot) on the "correct" strand you will yield a higher chance of cleavage. This can be a bit of a problem if you have repetitive regions, for instance.

This does not agree with our experience as well as literature, I am afraid. DNAse I preferentially digests in dependence on the sequence specific local structure, especially at sites adjacent to pyrmidines. See for instance: Nucleic Acids Res. 2002 December 15; 30(24): e139. Journal of Molecular Recognition Volume 7, Issue 2 , Pages 65 - 70 However you are right in so far that for small fragments enzymatic digests is probably the way to go. Alternative enzymes migh be CviJ1 (check Sambrook: "Molecular cloning" for more)

Is there a special reason why it has to be 500bp? Most shotgun libraries have inserts of around 1-4 kb. These can be easily achieved with several shearing methods. How large is your genome? The problem with DNase (or other enzymatic treatments) is that the resulting libraries are often non-randomly distributed...

Just a thought, is it possible that you mean meiosis? This would be true, of course.