Talk:Polymerase chain reaction

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Contents 1 Old comments 2 CATTAGAT 3 Har Gobind Khorana 4 "PCR reaction" 5 History of PCR 6 Taq is not a polymerase that is used always 7 Bicycle or Car? 8 elongation vs extension 9 micro-PCR 10 Could somebody please include Partial DNA match explanation in this article ? 11 possible rephrasing needed 12 ile code 13 Redirection 14 nomenclature 15 Is it a good example of PCR elecrtrophoregram 16 Use in identification of pathogens 17 GA Re-Review and In-line citations 18 Article is too big! 19 first image is not definitive 20 Restructuring article 21 Gene clonning 21.1 Changes have been made 22 PCR Station 23 LSD 24 Real-Time PCR 25 GA Review 26 Too technical for the general reader 27 PCR "Reaction" 28 PCR machine pics 29 Exponential applification 30 Lead picture change 31 AfD Polymerase Chain Reaction (simplified)

[edit] Old comments

There appears a concept called "Tm" in this article, but it is neither linked to an article with such title, or explained in this article itself. Please, define this term in its own article (and link to it), or define it here. Thank you. Pentalis 21 april 2:01 GMT -4:00 winter time.

• It stands for melting temperature, often abbreviated Tm in calculations. Not sure why it is Tm rather than Mt, though capital T usually stands for temperature. CheekyMonkey 12:49, 2 May 2005 (UTC)

• • Thank you very much, I also noticed you added the concept to the disambiguation page too, very kind of your part :-) Pentalis 6:25, 31 May 2005 (GMT -4:00 winter time)

Fixed to T_m. 203.218.136.155 14:39, 11 June 2006 (UTC)

In step 4 of figure 2 is not explained. How is the rest of the DNA past the primers truncated? All we see is a dotted line on the diagram suggesting it is cut somehow.

They don't get replicated, and hence drop out of significance entirely. (Among the millions of DNA molecules that eventually get made.) Its just a diagram to illustrate the concept, the actual reason is in the article. -- Natalinasmpf 13:27, 16 Jun 2005 (UTC)

[edit] CATTAGAT

There's a new program being released (under the GNU GPL) to perform primer specificity searches.

http://genome.cs.hi.is/cattagat/

If this looks useful to anyone then perhaps it can be added to the external links. I don't feel right doing it myself since I'm one of the authors of the software. - Haukurth 15:55, 21 July 2005 (UTC)

[edit] Har Gobind Khorana

I don't see how one can attribute the invention of PCR to Khorana. An invention (at least a patentable one) requires a reduction to practice, which he certainly never did. There is no reference cited for this either. In addition, the theromostabile polymerase was not known to Mullis at the time of his invention. Mullis got the Nobel Prize for the invention, not Khorana. I am therefore removing the attribution to Khorana.DonSiano 17:23, 22 August 2005 (UTC)

• Hi DonSiano. I left the actual reference to the paper (which you removed), as well as the actual text from the paper. In the paper, (and in lots of subsequent work), they used the procedure (which I quoted from the original paper) to construct genes in their study tRNA related genes. Some of the other references mentioned in the citations at the bottom of the article also mention Khorana's work. The actual text you removed is below:

• Much of the conceptual work on polymerase chain reaction was carried out in Har Gobind Khorana's lab at MIT ^[1]. Khorana and coworkers published a paper in 1971, describing repair replication and the process by which it could be used to synthetically replicate tRNA genes in E. coli. The major finding is given in this text:

The principles for extensive synthesis of the duplexed tRNA genes which emerge 
from the present work are the following. The DNA duplex would be denatured to 
form single strands. This denaturation step would be carried out in the presence of a 
sufficiently large excess of the two appropriate primers. Upon cooling, one would hope 
to obtain two structures, each containing the full length of the template strand 
appropriately complexed with the primer. DNA polymerase will be added to complete 
the process of repair replication. Two molecules of the original duplex should result. 
The whole cycle could be repeated, there being added every time a fresh dose of the 
enzyme. (Kleppe et al., 1971)

1. ^ [1]

• I actually redundantly reference the work. If you do not access to the reference, I would be happy to send it to you. In fact, if you read the paper, they actually reduce it to practice not only in the referenced paper, but in much of the subsequent work in synthesizing tRNA related genes. Regardless, the wording I used was being conservative and not attributing an invection to Khorana's lab, just that the conceptual work was laid down over a decade earlier. The quote that I use from the paper (12 years before the invention of PCR) is PCR, they just call it something different. In fact, in the referenced paper, they determine other conditions that are conducive to such amplification, such as minimal primer sizes. I could reference Khorana's subsequent work on amplifying various pieces of genes using the procedure. Granted, Khorana's group did not push automation, thermostable polymerases, et cetera, but it is no reason not to mention and reference the work.

I'll post the information back, unless you can explain to me why this work is not important to the history of PCR. If the wording seems controversial, maybe we could consider rewriting the section?\

--Skosuri 03:46, 9 June 2006 (UTC)

---

Hi Skosuri,

Well, PCR is one of those really big inventions that someone other than the almost universally acknowledged inventor thought of first. And the history of the invention is always more complicated than that which can be accommodated in a paragraph or two of an encyclopaedic article. Are there any inventions that don't have that characteristic? Both the courts (and presumably the nobel prize committee) considered this dispute and came down on the side of Mullis. There is no evidence that Mullis was aware of Khorana's work; and others certainly contributed to making it work: including, Henry Erlich, Norman Arnheim, Randall Saiki, Glen Horn, Corey Levenson, Steven Scharf, Fred Faloona and Tom White. And maybe even Kleppe has a claim too. There is, understandably, quite an impulse to knock the hero down a bit. The LSD ref is attributable to this tendency too, I think. I note that the "history" section of the article makes no mention of the others either. Nor is there any note taken that Mullis was a synthetic chemist put out of work by a machine to synthesize oligimers...

The fact is that PCR could have been invented years before Mullis came along, and no doubt a lot of people regret they missed it. One has to wonder, though, how many years would have passed before it was developed if Mullis hadn't done his bit...

The bottom line is, I don't think Khorana deserves all the space you would like to devote to him it this article, which would seem to outweigh Mullis' contribution. I certainly don't dispute that some mention might be made, but it is out of balance. Perhaps you could try some rewording of this: perhaps "like most inventions there are precursors and other actors than the one..." or something along those lines. Please don't just revert--let's try to reach some middle ground that is acceptable to us both.

There has been a lot of interest in PCR as a prototypical invention and it has been the subject of quite a bit of historical work, as I am sure you are aware. Perhaps what is really needed is a separate article on this, perhaps "PCR History"; I'd be willing to work on it with you--it could be interesting and worth doing. We could move a good bit of the history section over there and leave a ref to "main article" here. Which could shorten the PCR article which is already too long besides. What do you think? DonSiano 12:29, 9 June 2006 (UTC)

Call it History of polymerase chain reaction if you do it, to be consistent with other articles 203.218.136.155 14:39, 11 June 2006 (UTC)

[edit] "PCR reaction"

Is it considered acceptable to use the phrase "PCR reaction" or is this considered redundant? The phrase appears a couple of times in this article. (RAS syndrome and the associated discussion page provide an interesting treatment of the issue; a Google search for "'PCR reaction' and 'RAS syndrome'" also reveals some interesting hits).--GregRM 15:17, 21 December 2005 (UTC)

I try and remove the 'reaction' from the 'PCR reaction' whenever I can as it bugs me but maybe I'm just picky! I think it stems from the fact that acronyms such as PCR, ATM, CCTV and so on have become so commonplace it is no longer considered necessary to define them and so people lose sight of the actual meaning and consider the concept as a whole. Thus, when wanting to emphasise the fact that it is a reaction, the 'reaction' tagged onto the end. -luckyluke09

Yes it is redundant, always remove reaction coz this is an encyclopedia 203.218.136.155 14:39, 11 June 2006 (UTC)

Thanks for the replies...I removed references to "PCR reaction" yesterday.--GregRM 22:35, 12 June 2006 (UTC)

[edit] History of PCR

• History of PCR A great review article. Also PCR Protocols, PCR Bioinformatics, and discussion are included.

Links to other PCR topics removed as they are not informative for standard PCR, and should be under Real-time or quantitative PCR. The History of PCR link is much more informative for PCR, yet was removed by [user:Alan Au] -- Bioinformin

[edit] Taq is not a polymerase that is used always

I disagree strongly with the phrase that Taq is used in current practice. Taq is with current standards a bad polymerase and it is not necesary to use Taq. Taq has no proofreading activity and its processivity is not as good as other polymerases, so it is my opinion that wikipedia should take an independent stance and not link PCR to Taq

I also made a very imortant change. It is not nucleotides that are used in the reaction but deoxynucleotides-triphosphate

I think you mean deoxynucleoside-triphosphate. There is no such thing as a nucleotide-triphosphate, since the definition of a nucleotide is a nucleoside + phosphates. 130.243.248.239 14:29, 19 November 2006 (UTC)

Comment:

yes i completly agree to this. while taq polymerase certainly is the most famous and perhaps the one most used, it is far from the only one, nor is it one of the best polymerases available. an elucidation of the other available polymerases would be much appreciated. here assessing their properties, comparing price (relatively, not absolutely), areas of use. where it is appropriate to have a polymerase with high fidelity. 3' nucleases 5' nucleases. and so on and so forth.

thank you -broccolee

[edit] Bicycle or Car?

I was told that Mullis was driving his car and that he was going on vacation with his (first) wife and that he got the idea that he just had to try, so he turned the car around to the lab... Is the motorcycle source reliable?

Several theories abound, ranging from the drugs story to the possibility that Kary Mullis merely reduced to practice the idea that had been given to him by a co-worker who had attended a seminar discussing tRNA synthesis in a manner similar to what we now know as PCR. Another rumour is that Kary Mullis was not going to be named as an inventor on the patents (the glory instead passing to David Gelfand, a co-inventor on the Taq patents, and Randy Saiki), and came to work at Cetus one day with a gun, demanding to be named as the lead inventor. All rumour though, and not suitable for the main page. 159.92.30.11 12:53, 1 June 2006 (UTC)

[edit] elongation vs extension

i observe that one use the term elongation for the polymerase 'elongating' the primer along the dna strand. however the more correct term is extension. also elongation is used regarding protein synthesis.

the analogy is

melting-annealing-extension:PCR-cycle::intiation-elongation-termination:protein synthesis

i am fairly sure these terms are not used over one another. however you may know otherwise.

thats all.

[edit] micro-PCR

In my research on microelectromechanical systems (MEMS) devices, I ran across some information on micro-PCR devices, which are fabricated with MEMS techniques. They consist of arrays of reaction chambers with electric heaters, and their construction allows them to do faster PCR with smaller sample sizes (both DNA and reagents). Does anybody else know the history of micro-PCR (sometimes denoted μPCR) that can help me add some stuff on this topic? Good article: http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6TFC-4CPM20J-1-1&_cdi=5223&_user=1238599&_orig=search&_coverDate=01%2F15%2F2005&_qd=1&_sk=999799992&view=c&wchp=dGLbVlb-zSkWz&md5=ded7b4a9d79b6ef6bc883c7d08e00020&ie=/sdarticle.pdf

[edit] Could somebody please include Partial DNA match explanation in this article ?

I think partial DNA match is relevant topic to be included in this article. Could somebody please do that ? I need information about Partial DNA match interpretation. For example, it test is done on 13 alleles, and match is partial (on 10 of them), and reaction has not been succesful on 3 alleles: how should this be interpreted ? As far as I know, there are divided opinions in scientific community about this issue. Please help, this issue is important to me, and at the same time I think it is relevant to be included in this article, because it makes it more complete and understandable.

[edit] possible rephrasing needed

"Primers are short, artificial DNA strands — often not more than fifty and usually only 18 to 25 base pairs long nucleotides that are complementary to the beginning and end of the DNA fragment to be amplified." How can a nucleotide be 18-25 base pairs long, this needs to be rephrased.

removed nucleotide. 203.218.136.155 14:39, 11 June 2006 (UTC)

"Colony PCR - Bacterial clones (E.coli) can be screened for the correct ligation products. Selected colonies are picked with a sterile toothpick from an agarose plate and dabbed into the master mix or sterile water. Primers (and the master mix) are added - the PCR protocol has to be started with an extented time at 95^^C." Would you really use a sterile toothpick, Wouldn't a sterile wire loop be more appropriate? --KX36 10:47, 27 May 2006 (UTC)

[edit] ile code

Can someone explain why the DNA code for ile is AT(A/C/G) when the RNA code is AU(A/C/U)? I thought RNA is complementary to DNA so shouldn't the DNA be TA(A/G/T).

Explanation: If you recall, it is the antisense (noncoding) strand of the DNA that is used as the template strand for the mRNA, meaning that the actual coding DNA strand and the mRNA produced during transcription will be identical except for the G->U nucleotides. Here's a website with a few more diagrams to help: Transcription Amasa walker III 03:48, 23 June 2006 (UTC)

[edit] Redirection

Primer extension is not the PCR reaction! It's a method used in 5'termini mapping!

[edit] nomenclature

Replaced several descriptions "ionic" bonding with hydrogen bonding. Made some other changes. Am very new at Wikipedia editing but have been doing PCR and related technologies for many years. User:Twbeals31 26 July 2006

[edit] Is it a good example of PCR elecrtrophoregram

Why to put such a bad electrophoresis gel photo in the article? It is quite noisy and really bad. There are billions around for example mine. Does this have a some significance? --Araks 13:36, 16 September 2006 (UTC)

I agree. I work in a lab as well, so I can provide a better image.--Roland Deschain 18:41, 16 September 2006 (UTC)

Ok. I've added a gel from my own research. It's reasonably clean and produces one specific band for each reaction.--Roland Deschain 19:06, 16 September 2006 (UTC)

[edit] Use in identification of pathogens

There are several infectious diseases which require the use of PCR for identification of the causative agent as alternative techniques require incubation periods which render them useless. shouldnt this be added to uses?

[edit] GA Re-Review and In-line citations

Members of the Wikipedia:WikiProject Good articles are in the process of doing a re-review of current Good Article listings to ensure compliance with the standards of the Good Article Criteria. (Discussion of the changes and re-review can be found here). A significant change to the GA criteria is the mandatory use of some sort of in-line citation (In accordance to WP:CITE) to be used in order for an article to pass the verification and reference criteria. Currently this article does not include in-line citations. It is recommended that the article's editors take a look at the inclusion of in-line citations as well as how the article stacks up against the rest of the Good Article criteria. GA reviewers will give you at least a week's time from the date of this notice to work on the in-line citations before doing a full re-review and deciding if the article still merits being considered a Good Article or would need to be de-listed. If you have any questions, please don't hesitate to contact us on the Good Article project talk page or you may contact me personally. On behalf of the Good Articles Project, I want to thank you for all the time and effort that you have put into working on this article and improving the overall quality of the Wikipedia project. LuciferMorgan 02:17, 16 December 2006 (UTC)

[edit] Article is too big!

I propose moving the primer design information to Primer (molecular biology). I also propose creating a new page PCR optimization and moving all the PCR optimization information there as well. -Madeleine 00:46, 23 March 2007 (UTC)

[edit] first image is not definitive

the first image that's pcr tubes are not typical image for pcr. I am molecular microbiologist and never saw such colorful pcr mix. appealing image but not suitable I think. ArazZeynili 11:23, 21 April 2007 (UTC)

Neither have I--taqman probes have a red hue though, so it's possible that it's a fluorophore-based PCR. There's apparently some precipitate at the bottom of the tubes, lending credence to the fact that it's a colony PCR (which may have caused the coloration) as per the legend. It would be best to track the editor who inserted the pic to get more info. Malljaja 13:21, 21 April 2007 (UTC)

I had the same thought when I saw it added, but a quick googling for "colony pcr" found me this protocol which uses sucrose red in the mixture: http://www.cbs.umn.edu/~kclark/protocols/bactPCR.html. I've seen this before, in pre-made form -- dyes and some heavier stuff is added to the PCR mixture, this allows people to directly load the PCR solution onto a gel after the reaction is done, without taking an extra step to mix in loading buffer. (I didn't personally use it, someone else in the lab had bought one of these products.) Anyway, here's some examples of pre-made versions: [2], [3], [4].

Although the image wasn't typical, it wasn't technically wrong either. Not wanting to look a gift horse in the mouth, I didn't take issue with it at the time. One of us could certainly take a pic of a strip of PCR reactions next time we set one up and replace this pic, if you think the color is going to throw people off. I wonder if there should be a hand in it, for scale. -- Madeleine 14:40, 21 April 2007 (UTC)

I would put this 'gift' down in the article. person facing pcr for the first time will think that this is very typical pcr thing which is not. hand for scale goog idea ArazZeynili 22:52, 21 April 2007 (UTC)

I've replaced the picture with one of my own, a strip of eight 100ul PCR reactions I set up. Madeleine 15:55, 14 May 2007 (UTC)

[edit] Restructuring article

I think the history should go higher, but after the basic description of PCR. What do people think about rearranging the article into this form:

• The polymerase chain reaction (description of basic protocol/process)

• sample procedure

• History of PCR

• patent wars

• Applications of PCR
• PCR optimization and variations (many modifications, eg hot-start PCR, have been developed as optimizations, so I think the two subsections belong together)

• optimization
• modifications to the technique

I'm open to different orderings, of course. Mainly I'd like to move the optimization and variations away from the core "this is how PCR works" section. Madeleine 19:08, 19 May 2007 (UTC)

Hi Madeleine, I do not feel very strongly about any re-arrangements. To me it seems that the practical parts almost deserve their own articles with "PCR" covering the basic principle, application, and history. The major shortcomings of the article were convoluted and redundant text and misconceptions creating mystification, which thankfully have largely disappeared, but may crop up again every now and then. I'd suggest you go on with any changes you feel are necessary--I'll offer any suggestions if necessary and time permitting. Many thanks! Malljaja 19:07, 20 May 2007 (UTC)

[edit] Gene clonning

The gene clonning section in this article is of no relevance, as far as I can see. PCR can indeed be used for detection of genetic modifications, but anyways, the entry does'nt seem to spell out anything of that sort. ώЇЌĩ Ѕαи Яоzε ^†αLҝ 23:46, 11 June 2007 (UTC)

Using PCR for gene cloning (or cloning of any piece of DNA for that matter) is one of the most common methods used in a molecular biology lab nowadays; so it's extremely relevant and really belongs here!!! To improve the section on PCR-based gene cloning, it should be expanded to include methods such as TA-cloning, overlap-PCR ("PCR splicing"), linking to nested PCR, and highlighting of potential pitfalls (cloning of long DNA fragments, proofreading, etc, much of which is already alluded to in other sections). I'll try to get to that soon, but anyone familiar with the techniques is encouraged to chip in! Malljaja 08:57, 12 June 2007 (UTC)

I guess just because TA cloning protocol uses treminal transferase property of Taq we can't use it over here. Indeed Taq is an enzyme used in PCR but exploiting the property is just an ease of clonning PCR products directly which is gained by using special vectors rather than special PCR protocols.

TA clonning is a protocol used to clone genes, but its not a PCR procedure used in cloning per se. It is more to do with the property of Taq (and similar DNA polymerases) rather than the PCR protocol. If we start including everything that uses PCR or PCR products then we will end up with the largest entry on wikipedia. In the latter case we ill have to include DOT BLOTS, SUBSTRACTIVE HYBRIDISATION, and even TRANSCRIPTOMICS!!! ώЇЌĩ Ѕαи Яоzε ^†αLҝ 09:37, 12 June 2007 (UTC)

PCR is an extensively used technique permeating through molecular biology like water through cloth, and the length of the article simply reflects that--as long as the PCR principles and application are conveyed in a straightforward non-redundant prose (something that has improved only recently), I do not see any problem with it (the index at the top allows easy jumping to the relevant sections). Moreover, there's been discussion about moving sections out of the main article, which has happened, e.g. with PCR optimization, which is a good way to deal with methods that require more detailed description and deserve their own article. Nonetheless, PCR-based cloning needs to be in the main article, perhaps broken down to nuts and bolts (if given in more detail elsewhere). TA cloning is indisputably based on PCR (as you yourself pointed out), and should be dealt with as such.Malljaja 11:07, 12 June 2007 (UTC)

Sorry, let me not be considered rude. I think we will leave it for others to comment. My point is, clonning is not a PCR protocol. Moreover the article entry on clonning doesn't spell out anything about TA or the terminal transferase property of Taq. May be if it does we can decide on if it fits in or not. On a lighter note, I do TOPO quite regularly and I have started hating minipreps. ώЇЌĩ Ѕαи Яоzε ^†αLҝ 11:13, 12 June 2007 (UTC)

Thanks for clarifying--now I see what you're getting at. I completely agree with you that the section on PCR-cloning contains precious little about the properties of Taq and how they are deployed in PCR-cloning techniques, and I think those belong in there for completeness (see my earlier comment). So I'd be happy to see such info in this section--if need be, PCR cloning would be seeded as a new article, and only referenced in the main article. Yes, may be others have thoughts on that as well. TOPO gave me some headaches in the past, so I've been doing most of my cloning by PCR (incidentally!). Malljaja 12:48, 12 June 2007 (UTC)

Most of these "uses" of PCR listed here are not PCR protocols, it's not just the "cloning". Many of the methods are here for the same reason cloning is listed -- they greatly benefit on PCR's ability to isolate a particular genomic region from whole genomic material. (I think this cloning word is confusing, it seems to refer to recombinant DNA / transgenic usage, which does greatly depend on isolating a particular region, not so much about isolating clonal DNA sequences.) All of these techniques (listed here) could be done without PCR but now use PCR to rapidly isolate the desired region:

• genetic fingerprinting

• paternity testing

• (sequencing)

• detection of diseases
• genotyping of specific mutations

• "cloning" (gene isolation -> recombinant DNA)

I've clustered these, they look like they could be condensed, and all of them could go under a single subject eg, "PCR is widely used as a tool for isolating and amplifying a particular region from whole genomic sample. Although it is possible to isolate fragments without PCR, this ability of PCR has made some techniques much easier to perform and therefore enabled their widespread usage."

Another major usage of PCR would be amplification of very low copy number -- although there's non-PCR methods of amplification that have less bias (ie. RCA methods). This overlaps with forensic usage (DNA fingerprinting is doable without PCR, but PCR enables genetic analysis for many samples that wouldn't otherwise be analyzable). The following arguably depend on this ability:

• (viral DNA detection)

• AIDS testing

• analysis of ancient DNA

The only remaining "use" here is the "comparison of gene expression" using quantitative PCR. Of course, we could always just hybridize unamplified cDNA to a microarray and never use PCR, right?

I think the entire section could needs to be reorganized, possibly under these major uses for PCR -- "isolation of genomic regions", "amplification of small amounts of DNA", "quantitative PCR". What do people think about this? In the meantime, I'm off to do some colony PCRs off my TOPO TA cloned bacteria.  ;-) -- Madeleine 17:35, 12 June 2007 (UTC)

I reckon Applications of PCR should be one single section. Types of PCR protcols should be expanded extensively either here, or seperate entries. Moreover as I said earlier I dont see why entries were added without citations. Anyways, I think this article on the whole needs some copy editing. If there are others who feel the same, please express your views. Because many students do refer to wikipedia for basic knowledge on things like PCR and it needs to be clear, to the point and with proper citations. Cheers ώЇЌĩ Ѕαи Яоzε ^†αLҝ 20:40, 12 June 2007 (UTC)

I can't tell whether you're agreeing with my proposal or not. -- Madeleine 20:54, 12 June 2007 (UTC)

I like Madprime's suggestions of re-organizing the article where necessary--I think she's already done a great job in recent edits (the article has been in much worse shape just a couple of months ago and has much improved since), so I trust that any changes she'll deem necessary will be sound ones. Wikiality you're preaching to the converted, I also think the article is lacking enough citations, and this small number of citations was a major reason for a recent downgrade in a Wiki review. Rather than lamenting the lack of citations, however, energy might be better spend finding and inserting references, something you're more than welcome to do (it's on my to-do list as well). Malljaja 21:32, 12 June 2007 (UTC)

Sorry Malljaja, I think this is getting an unfortunate personal U turn. I will exit here if thats the case. Let me make it clear. PCR is not a clonning protocol. Madeleine agrees to that too. I bumped into this article last night when I was editing (including adding reference) to GC-content article. You can check the article for the changes I have made. Anyways, my point is, if no one is fighting, that the applications should be in one single section, and doesn't need much explanation for each of them. I think we can all agree on one point, that PCR can be used as a first step for loads of applications. Just because PCR is used as the first step leading to something that can't be called as a seperate PCR protocol, same as isolation of DNA. Just because DNA needs to be isolated as a first step that doesn't mean that all the applications would be explained in details under Isolation of DNA. If we agree on this it would be nice. I appreciate the efforts taken by Mandeline and all others in fixing the article, but I think we still need citations for many things stated here. Anyways, I write in wikipedia to share my years of research experience, but not to be criticised for preaching. Sorry if I was anoying, I think I will leave this talk page to save my own dignity. Thanks ώЇЌĩ Ѕαи Яоzε ^†αLҝ 21:45, 12 June 2007 (UTC)

Just to add one more thing, citation is requested not because others cant find it, but because it is easier for the ones who wrote it to cite, because after all they would have read it somewhere. ώЇЌĩ Ѕαи Яоzε ^†αLҝ 21:52, 12 June 2007 (UTC)

[edit] Changes have been made

I've rearranged things quite a bit. Some references have been added, in the process, but please appreciate that adding references for already existing material is pretty tedious.

The list of PCR variations has been moved to the bottom, since it's pretty long and lacks narrative order. It may be possible to cluster it a bit. Some are PCR optimization, meant to reduce amplification of incorrect product (Hot-start PCR, Nested PCR, touchdown PCR). Methylation-specific PCR is related to allele-specific PCR (it's basically an allele-specific PCR where the "allele" is a uracil vs. methylcytosine). Both inverse PCR and TAIL-PCR are used to isolate unknown sequence next to known sequence (as I understand it)... but TAIL-PCR is also somewhat like nested PCR... -- Madeleine 04:53, 13 June 2007 (UTC)

[edit] PCR Station

The premier site for researchers conducting PCR, helped me quite a bit during my Ph.D.

PCR —Preceding unsigned comment added by Testtubeboy (talk • contribs) 10:10, 2 July 2007 (UTC)

Going by Wikipedia's external links guidelines ... This site appears to aggregate paper abstracts and tacks on some advertising links. It is far from being "accessible to the reader", and it contains significant amounts of advertising. The claim that it helped you quite a bit during your PhD is actually an argument against its inclusion: Wikipedia is not a manual, guidebook, or textbook, there is a strong argument against providing or linking to content intended to aid use of PCR protocols in research.

The external links section of wikipedia pages often degenerates into a free-for-all advertising section for various links related to the subject of the page. Wikipedia is not an advertising space. For that reason I've tried to clean up the link section and trim it down to a core of links significantly useful additional content to readers. The PCR Station link looks like this sort of low-content advertising to me. In addition, I distrust your intentions in adding this link; you have made no contributions to wikipedia other than trying to add this link. Madeleine 15:11, 2 July 2007 (UTC)

[edit] LSD

• Better reference for Kary Mullis's LSD use

Is his own autobiography, Dancing Naked in the Mind Field. Here is a NYTimes review of the autobiography that mentions the LSD use and a site that has copied an excerpt from the book. http://www.csp.org/chrestomathy/dancing_naked.html http://www.nytimes.com/books/98/10/11/reviews/981011.11teresit.html Kevin143 08:47, 22 September 2007 (UTC)

[edit] Real-Time PCR

I suggest an addition to the "Variations on the basic PCR technique" section as follows:

RT-PCR: (Real-Time PCR) is a powerful and rapid technique for nucleic acid amplification. The accumulation of specific products in a reaction is monitored continuously during cycling. This is usually achieved by monitoring changes in fluorescence within the PCR tube. Touchstone42 13:50, 15 October 2007 (UTC)

Real-time PCR is already among the different techniques (under quantitative PCR) that appear under "Variations..." Have a look how it fits in there. If you talk about fluorescence, it needs to be specified from where the fluorescence originates. Also, its main use is quantification; your lead sentence basically describes PCR and doesn't mention quantification. Please note that RT-PCR is the common acronym for reverse-transcription PCR, so it would be inappropriate to use it here (qPCR or qRT-PCR (for quantitative reverse-transcription PCR) are the most commonly used abbreviations). Malljaja 15:31, 15 October 2007 (UTC)

I agree with everything you say. RT-PCR is the common acronym for reverse-transcription PCR however it is also a very common acronym for real-time PCR. I have found RT-PCR in many many places referring to real-time PCR. I think this needs to be accepted. Also, (I may be wrong) but quantitative PCR does not have to be real-time and real-time PCR does not have to be quantitative so I don't think the two terms are interchangeable. Touchstone42 15:31, 16 October 2007 (UTC)

I've likewise seen real-time PCR abbreviated as RT PCR in the literature. Some researchers have even dubbed reverse-transcription real-time PCR as RT-RT PCR, which sounds a wee bit like a stutter though. Ultimately, context is important, and acronyms often follow local conventions in the literature (i.e. full name followed by a, sometimes whimsical, acronym in parenthesis). However, as this entry gives a comprehensive overview of PCR and its different twigs and branches, simply calling real-time PCR "RT PCR" would be inappropriate, as it collides with "RT PCR" as used for reverse-transcription PCR. "Variations..." mentions the different naming conventions and conflicts, and it also touches on the issue that quantitative PCR methods are not always based on real-time PCR. Perhaps this section can be improved so that some of the naming and other issues can be put to bed. Malljaja 16:27, 16 October 2007 (UTC)

[edit] GA Review

This article does not meet the Good Article criteria at this time. Please see this page for the complete review. Dr. Cash 06:49, 23 October 2007 (UTC)

I've had a first stab at improving the article, mainly copy editing (i.e., boldly paring back unnecessary and convoluted wording), and inserting of refs as per the review. I've not done much work on the "History" section yet. This entry has slowly been improving, but it still needs come cleaning up--its previous length seemed to have invited some redundancy, manifested as overlinking and lack of reference to other sections, and some disorganization to creep in. Making it more succinct might help keep it more stable and to the point. Some of the the figures (Fig 3 in particular) look fairly poorly, and the text needs some more polishing to make it more accessible to the layman audience. I'm going to leave it for now, to give others the opportunity to chip in where they see fit. Malljaja 16:19, 23 October 2007 (UTC)

[edit] Too technical for the general reader

I agree that the article is too long, however I feel there's a bigger problem.

The article is much too technical for the average reader. There are far too many scientific terms. Reading the article I found myself quickly needing to either follow the links or grab a dictionary.

A wikipedia article shouldn't require additional reference materials in order for the general reader to gain a grasp of the basics of PCR (or any other topic for that matter; I could easily write an article on any number of Roman Emperors that would leave the general reader feeling just as out of depth as I do with this one).

In my opinion this article needs a rewrite to make it more accessible for readers not intimately familiar with molecular biology and related disciplines.

This article Kinetic energy penetrator on a ballistic technology does a much better job, again in my opinion, in explaining the subject without a blizzard of recondite terminology.

Therefore I've added the "cleanup-jargon" tag.

PainMan 08:40, 28 October 2007 (UTC)

I agree with you that there's a fair amount of specialized language in the article. It would help however if you were more specific--which sections are in your opinion most affected? I had a look with an eye on jargon, and the section that I see most plagued by it is the "Uses of PCR" section. Unfortunately the article you chose as a positive example (the KEP) lacks citations, raising the question of how much of the true complexity of the subject it reflects. Similar to other areas in the natural sciences--quantum mechanics come to mind (see e.g., Quantum computer, which if scaled to weather phenomena represents a class 5 hurricane in terms of arcane terminology)--articles dealing with molecular biology may not unlock themselves on first reading, and likely require some additional reading in other related entries. Sequence alignment is a featured article covering a fairly complex subject, so perhaps it could be the guiding light. Malljaja 12:17, 28 October 2007 (UTC)

[edit] PCR "Reaction"

Since the 'R' in PCR is for the word 'Reaction' its a tautology to say PCR Reaction. This means Polymerase Chain Reaction Reaction. I suggest the removal of all such occurrence within the text. —Preceding unsigned comment added by 129.215.149.97 (talk) 23:38, 9 March 2008 (UTC)

Agreed. You're most welcome to be bold and remove them yourself. Adrian J. Hunter^{(talk•contribs)} 02:14, 10 March 2008 (UTC)

I've gone through and changed the ones I found. Notably, sometimes "PCR reaction" is used to refer to the mixture in the tube and in some cases it I think it's preferable to say "PCR mixture" rather than simply saying "PCR" (which refers more to the process than the mixture in the tube). Madeleine ✉ ✍ 03:05, 10 March 2008 (UTC)

[edit] PCR machine pics

Wow, those are impressive! Very cool images of old machines. I think maybe they should go in the history section though. PaleWhaleGail also added a pic of a modern machine to the thermal cycler page, I think that would be more appropriate to the principle/procedure section. Madeleine ✉ ✍ 03:15, 14 March 2008 (UTC)

I'm glad you like the picture of the old machine. I've seen a few of them on the way to the skip. Young ones are shocked when they realise what it is. I wonder what they'd think of having to add kelnow fragment every cycle! If you want to put the three water bath machine in the history section, i think it might work well. Agesworth (talk) 08:14, 2 April 2008 (UTC)

[edit] Exponential applification

A see some changes based on the question of whether PCR produces exponential amplification. I believe it does, by most methods. An exception is the QuikChange method, where amplification is linear, as the extended primers don't overlap. QuikChange is limited by either the number of templates, or the number of primers.

Question. Does the QuikChange method correspond to one of the "Variations on the basic PCR technique"? Agesworth (talk) 08:14, 2 April 2008 (UTC)

Can you provide a link to a description of QuikChange PCR? My initial googling indicates this is a mutagenesis kit; while it uses PCR to generate the target fragment I can't see any indications that this is different from a normal PCR. Maybe it's tagged PCR? (That doesn't seem to be on the list; could be added.) But this would still be exponential. Madeleine ✉ ✍ 12:47, 2 April 2008 (UTC)

Yes, QuikChange is a commercial mutagenesis kit, but the concept and method is so simple that you certainly don’t buy “the kit” more than once. It is also known as “whole plasmid PCR” (The earliest reference I have is: Anne Hemsley, Norman Arnheim, Michael Dennis Toney, Gino Cortopassi, and David J. Galas A simple method for site-directed mutagenesis using the polymerase chain reaction Nucl. Acids Res. (1989) 17: 6545-6551; doi:10.1093/nar/17.16.6545). Originally they used Taq, but now we use Pfu.

Your two primers are substantially complementary. If primer pairs anneal, there is pretty much nothing to extend. Where a primer anneals to the plasmid, it is extended all the way around until the polymerase reaches the 5’ end of the primer, where polymerisation stops. It has replicated one strand of the plasmid, with a remaining nick, and perhaps a missing base or two at the nick. The reverse primer does the same thing, except that the nick is in a different place, 30-40 basepairs away. When the two strands anneal together, you have a doubly nicked plasmid, but the overhang is so large that it is very stable, transforms well, and the two nicks are repaired by the cell’s repair DNA repair mechanisms. Thus, the method is also an example ligation free subcloning.

As the primer extensions don’t contain sequence complementary to the other primer, primers can’t anneal to primer extensions. Polymerisation can only occur when a primer anneals to the plasmid template. The reaction is therefore not exponential/geometric, but linear. Compared to normal PCR, the amount of template used is large, and the number of cycles is small.

All of this is detailed in the product description, eg http://www.chemistry.montana.edu/~martint/BCHM444/QuickChange.Mutagenesis.pdf (a google result).

What’s not clear to me is whether QuikChange is really “PCR”. It uses a PCR machine, and all of the standard reagents and methods, but as I said, primers don’t anneal to primer extensions, and so the reaction is not actually a “chain” reaction. I don’t know what “tagged PCR” is. Agesworth 23:51, 2 April 2008 (UTC)

Tagged PCR is different, it's simply adding additional sequence to the 5' end of primers; these sequences can be used for a later amplification from the added sequence, or whatever else you want...

I think you're right, I wouldn't describe this as PCR... it's not even intended as DNA amplification, really. Other DNA amplification strategies that aren't PCR are rolling circle (also linear) and exponential/hyperbranched rolling circle amplification. (Interestingly, these all involve circular templates.) I can't think what article this would be appropriate to, but it's so specific it doesn't seem important to cover it.Madeleine ✉ ✍ 00:05, 3 April 2008 (UTC)

[edit] Lead picture change

The new pic is hard to read and is copyrighted-- and this is is not fair use. I've got an obvious bias for thinking so, but I thought the old pic was fine. I was pleased to see it used elsewhere: [5] -- Madeleine ✉ ✍ 00:41, 8 April 2008 (UTC)

I don't know your bias, but I agree, the picture was not suitable for multiple reasons. --Agesworth 02:07, 8 April 2008 (UTC) —Preceding unsigned comment added by Agesworth (talk • contribs)

It's my hand. :-) Madeleine ✉ ✍ 03:56, 8 April 2008 (UTC)

If the pic is indeed copyrighted it preempts any rationale for use here, and I agree that it's not very appealing to the eye. It would be nice though to have a pictorial summary diagram of the PCR process, especially alongside the lead. The "hand" picture could still be in there, though ;-). Malljaja (talk) 10:10, 8 April 2008 (UTC)

Well, I made that PCR diagram (figure 2) farther down in the page. If it could be improved at all just let me know. I think it's too big for the lead, though. I could try making a version of that that only depicts the double-stranded molecules after each step, which would make it a bit smaller. Point out a link if you see a pic you like online that would be good.

Would you worry about such a pic being redundant with the more detailed version fig 2? Madeleine ✉ ✍ 13:21, 8 April 2008 (UTC)

The new and now deleted pic encapsulated the basic principle of PCR well, in that it visually connected the DNA double helix with the PCR process, which is great for a layperson audience. Figure 2 is very good too, but it requires some knowledge to fully get one's head around it--eg there's no explanation as to what the individual bars and errors represent. A more detailed legend would be starting point, but a linkage with the structure of DNA that now everyone recognises would really top it off. So a fleshed-out version of figure 2 would be excellent for the lead. So that would be my 2 cs. I don't want to foist this on you though--if you have time (and drawing tools) to redo the figure that would be great, otherwise we do it perhaps at small increments. Thanks!Malljaja (talk) 14:37, 8 April 2008 (UTC)

Bars? Errors? The picture has elements in it not explained in the text that seem important but could be removed: the polymerase (green circle) is depicted in the first annealing and extension steps, the template DNA is blue, the primers are red, and newly synthesized DNA is green. Because this is an SVG, it's fairly easy to delete things or change the color of things. Which, if any, of these items should be removed?

It sounds like you'd like something at the top that depicts the unraveling of a DNA double helix to two lines, to explain that the lines are representing paired DNA strands. This could be added at the top, maybe.

I think a PCR diagram is necessarily information dense. Note that the copyrighted image that started this discussion is so dense that it would take up the entire width of the page to become readable. To avoid this level of density I had avoided trying to labeling everything in the diagram I made. It also inherits a style from the diagram I was copying/correcting [6]. Madeleine ✉ ✍ 15:56, 8 April 2008 (UTC)

My apologies, that should have been "lines and arrows"--I've been working too much on graphs these days... I'd expect a general audience might be a little flummoxed by the lines and arrows in much the same way as I am, when I'm looking at some arcane chemical drawings. So how about expanding the legend by including what the lines, circles, etc represent? I agree though that it's going to be a little bit of a challenge to accommodate all the other details I suggested. The DNA helix I envision would be rather small. I'm rather hamfisted when it come to drawing, so I'm not going to touch it myself for now. Malljaja (talk) 16:15, 8 April 2008 (UTC)

[edit] AfD Polymerase Chain Reaction (simplified)

There is currently a simplified version of this article, Polymerase Chain Reaction (simplified), which is being considered for deletion at Wikipedia:Articles for deletion/Polymerase Chain Reaction (simplified). - Eldereft ~(s)talk~ 22:25, 5 May 2008 (UTC)

Closed - deleted as unnecessary, at least for the nonce. - Eldereft ~(s)talk~ 07:29, 11 May 2008 (UTC)