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Odd point mutations


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#1 fishdoc

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Posted 13 August 2009 - 12:35 PM

Lately I've been doing a lot of cloning into pBluescript and some derivatives made in our lab. The plasmid is then going into XL1-Blue MRF' cells by electroporation. This has been a successful and efficient strategy in the past, but lately, sequencing of the plasmid constructs has shown a seemingly inordinate amount of point mutations in the sequence.

I originally asked a question regarding this a couple months ago, thinking it may have had something to do with using a ligation reaction product as template, and perhaps that had an effect on the fidelity of Phusion. However, sequencing the DNA that was used for ligation into pBluescript showed no mutations from amplification using Phusion, so the point mutations (there were 4 total in a 2 kb sequence) all occurred somewhere between ligation into pBluescript and a plasmid prep out of the E. coli strain. I have a hard time believing the mutations are being introduced by E. coli, but that seems to be where the mutations are occurring.

I tossed out the original construct described above and remade it from scratch. Upon insertion into pBluescript and sequencing, I again found 2 colonies with the insert and sequenced the plasmid. One of them was fine, the other again had some point mutations.

At this point, I figured maybe it was just the gene I was inserting, and maybe it was an isolated thing.

However, within the past couple weeks, I've been doing some more cloning and have seen a few more of these point mutations pop up, again in pBluescript in XL1-Blue MRF'. I have not yet gotten to sequencing the PCR products used for cloning, so it is possible that they are the source of the mutation, but based on my experience with the stuff above, I think it's probably not from the polymerase. I had 4 clones from a construct I'm making, and each one of them had a different point mutation.

Most of the mutations are G>T or C>A, but I noticed a couple today were C>T.

The really odd thing is that I'm doing different constructs simultaneously (basically I'm flag-tagging a bunch of bacterial proteins), and 3 of the other fusions I'm working on cloned fine, and their sequencing is perfect. That leads me to believe it's not the polymerase, and it's not any sort of mutagen contamination in any of the chemicals or media that I'm using, because they're all being made with the same reagents and grown in/on the same media.


Any ideas on what MIGHT be going on? I'm going to go back right now and compare the mutated to the non-mutated ones to see if any were gel-purified as opposed to PCR purified (UV damage). I really can't think of much else that it could be. Could the cells have lost their error-proofing while in the freezer? I don't know of any times where our stock cultures would have thawed, or if anyone would've let them thaw while using them.

#2 Teagan27

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Posted 15 August 2009 - 08:31 PM

Lately I've been doing a lot of cloning into pBluescript and some derivatives made in our lab. The plasmid is then going into XL1-Blue MRF' cells by electroporation. This has been a successful and efficient strategy in the past, but lately, sequencing of the plasmid constructs has shown a seemingly inordinate amount of point mutations in the sequence.

I originally asked a question regarding this a couple months ago, thinking it may have had something to do with using a ligation reaction product as template, and perhaps that had an effect on the fidelity of Phusion. However, sequencing the DNA that was used for ligation into pBluescript showed no mutations from amplification using Phusion, so the point mutations (there were 4 total in a 2 kb sequence) all occurred somewhere between ligation into pBluescript and a plasmid prep out of the E. coli strain. I have a hard time believing the mutations are being introduced by E. coli, but that seems to be where the mutations are occurring.

I tossed out the original construct described above and remade it from scratch. Upon insertion into pBluescript and sequencing, I again found 2 colonies with the insert and sequenced the plasmid. One of them was fine, the other again had some point mutations.

At this point, I figured maybe it was just the gene I was inserting, and maybe it was an isolated thing.

However, within the past couple weeks, I've been doing some more cloning and have seen a few more of these point mutations pop up, again in pBluescript in XL1-Blue MRF'. I have not yet gotten to sequencing the PCR products used for cloning, so it is possible that they are the source of the mutation, but based on my experience with the stuff above, I think it's probably not from the polymerase. I had 4 clones from a construct I'm making, and each one of them had a different point mutation.

Most of the mutations are G>T or C>A, but I noticed a couple today were C>T.

The really odd thing is that I'm doing different constructs simultaneously (basically I'm flag-tagging a bunch of bacterial proteins), and 3 of the other fusions I'm working on cloned fine, and their sequencing is perfect. That leads me to believe it's not the polymerase, and it's not any sort of mutagen contamination in any of the chemicals or media that I'm using, because they're all being made with the same reagents and grown in/on the same media.


Any ideas on what MIGHT be going on? I'm going to go back right now and compare the mutated to the non-mutated ones to see if any were gel-purified as opposed to PCR purified (UV damage). I really can't think of much else that it could be. Could the cells have lost their error-proofing while in the freezer? I don't know of any times where our stock cultures would have thawed, or if anyone would've let them thaw while using them.


Hi,

Which polymerase are you using? I had 5 point mutations in my insert because i was using a non-cloning grade polymerase.. yet a fellow student of mine had perfect sequence using the same enzyme? Ever thought of using Blue-light (SYBR safe) instead of UV when gel purifying?

Cheers

#3 phage434

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Posted 16 August 2009 - 06:07 AM

Sequencing PCR products directly prior to cloning will never show the accuracy of the PCR. You must clone the fragments, and sequence individual clones. Cloning selects a single molecule, which may have errors. Sequencing from a PCR reaction provides the average sequence of a very large population of DNA fragments. The errors are happening in your PCR reaction; you just cannot see them by sequencing the result.

#4 eldon

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Posted 16 August 2009 - 07:08 AM

Sequencing PCR products directly prior to cloning will never show the accuracy of the PCR. You must clone the fragments, and sequence individual clones. Cloning selects a single molecule, which may have errors. Sequencing from a PCR reaction provides the average sequence of a very large population of DNA fragments. The errors are happening in your PCR reaction; you just cannot see them by sequencing the result.


I agree with this. It is the most likely scenario. However, are you cloning from the original bacterial source or have they been propagated in the lab for too long such that point mutations might occur? This is a long shot, but something to think about when maintaining strains.

#5 fishdoc

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Posted 16 August 2009 - 07:28 AM

Lately I've been doing a lot of cloning into pBluescript and some derivatives made in our lab. The plasmid is then going into XL1-Blue MRF' cells by electroporation. This has been a successful and efficient strategy in the past, but lately, sequencing of the plasmid constructs has shown a seemingly inordinate amount of point mutations in the sequence.

I originally asked a question regarding this a couple months ago, thinking it may have had something to do with using a ligation reaction product as template, and perhaps that had an effect on the fidelity of Phusion. However, sequencing the DNA that was used for ligation into pBluescript showed no mutations from amplification using Phusion, so the point mutations (there were 4 total in a 2 kb sequence) all occurred somewhere between ligation into pBluescript and a plasmid prep out of the E. coli strain. I have a hard time believing the mutations are being introduced by E. coli, but that seems to be where the mutations are occurring.

I tossed out the original construct described above and remade it from scratch. Upon insertion into pBluescript and sequencing, I again found 2 colonies with the insert and sequenced the plasmid. One of them was fine, the other again had some point mutations.

At this point, I figured maybe it was just the gene I was inserting, and maybe it was an isolated thing.

However, within the past couple weeks, I've been doing some more cloning and have seen a few more of these point mutations pop up, again in pBluescript in XL1-Blue MRF'. I have not yet gotten to sequencing the PCR products used for cloning, so it is possible that they are the source of the mutation, but based on my experience with the stuff above, I think it's probably not from the polymerase. I had 4 clones from a construct I'm making, and each one of them had a different point mutation.

Most of the mutations are G>T or C>A, but I noticed a couple today were C>T.

The really odd thing is that I'm doing different constructs simultaneously (basically I'm flag-tagging a bunch of bacterial proteins), and 3 of the other fusions I'm working on cloned fine, and their sequencing is perfect. That leads me to believe it's not the polymerase, and it's not any sort of mutagen contamination in any of the chemicals or media that I'm using, because they're all being made with the same reagents and grown in/on the same media.


Any ideas on what MIGHT be going on? I'm going to go back right now and compare the mutated to the non-mutated ones to see if any were gel-purified as opposed to PCR purified (UV damage). I really can't think of much else that it could be. Could the cells have lost their error-proofing while in the freezer? I don't know of any times where our stock cultures would have thawed, or if anyone would've let them thaw while using them.


Hi,

Which polymerase are you using? I had 5 point mutations in my insert because i was using a non-cloning grade polymerase.. yet a fellow student of mine had perfect sequence using the same enzyme? Ever thought of using Blue-light (SYBR safe) instead of UV when gel purifying?

Cheers


Phusion from NEB. I have thought it could be the UV, but if that were the case, wouldn't my sequencing of the gel purified product show the mutations?


edit: nevermind, I see where it could still be from the PCR product and the sequencing not picking it up. I will go back in my notebook and compare sequences that were gel purified as opposed to purified right from the PCR reaction to see if I can find any correlation to more mutations in the UV exposed samples.

Edited by fishdoc, 16 August 2009 - 07:32 AM.


#6 fishdoc

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Posted 16 August 2009 - 07:30 AM

Sequencing PCR products directly prior to cloning will never show the accuracy of the PCR. You must clone the fragments, and sequence individual clones. Cloning selects a single molecule, which may have errors. Sequencing from a PCR reaction provides the average sequence of a very large population of DNA fragments. The errors are happening in your PCR reaction; you just cannot see them by sequencing the result.




I had considered that option, too, but wasn't sure it was a real possibility or not.

#7 fishdoc

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Posted 16 August 2009 - 03:23 PM

I went back in lab notebooks and compared clones using gel purified DNA to PCR purified DNA. Of 4 constructs made using DNA from gel purifications, 3 of the 4 had perfect sequences following cloning. The 4th had 3 isolates with an insert, two of which had point mutations. Of those 2, 1 of them had 4 mutations in a 2 kb sequence.

Two other clones using PCR purified DNA had 1 with a correct sequence, and another that had point mutations. Of the 4 isolates of the 2nd clone, each one of them had a single point mutation, all at different positions throughout the sequence. This insert was about 1.2 kb.

So in total, I've made 6 constructs recently, 4 using gel purified DNA and 2 using PCR purified DNA. Five of these originated as PCR products; gel purification was required for a couple because of some nonspecific amplification. Of the 6 constructs, two of them had multiple point mutations, but apparently it's not related to gel purification (UV), because it's happening with the PCR purifications not associated with UV exposure.

So I guess it's either in the bacteria after cloning, or it's a product of the PCR reaction. But as I said above, most of the cloning I've done that results in an insert originates from PCR reactions, and not all clones end up with any mutations. So I wonder why it would happen with some products, but not others. All reactions are done using the same conditions (other than annealing temps) with Phusion polymerase. I received the suggestion when I originally posted this problem that using DMSO (1.5% per reaction) could cause a problem, and maybe that's true. I haven't removed that from my reactions, but I think I will when I go back to amplify other DNA. In any case, however, it can't be a certainty that the DMSO is causing the mutations, because other products work just fine.

This is really odd, at least to me.


As for the question regarding the source of the template DNA, it's from a genomic DNA prep. The original source of that prep is a frozen stock. The genome of the bacterium has been sequenced. Had these mutations been consistently in one place in regards to the genetic sequence, I would think it possible that it may just be a change in the sequence from one strain to another or something like that... or too much time in the freezer. But seeing as the clones are having different mutations in different positions, I don't think it's due to the DNA template having mutations, but rather the polymerase is making mistakes (not likely, IMO) or the E. coli strain is making mistakes (even more not likely).

#8 HomeBrew

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Posted 16 August 2009 - 05:19 PM

My next step would be to switch to another high fidelity polymerase and see if the situation improves. You could also try doing fewer cycles, or drop your extension temp a bit, or try another E. coli recipient strain...

But, a further question -- why do you need more than one mistake-free clone?

#9 fishdoc

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Posted 16 August 2009 - 06:02 PM

My next step would be to switch to another high fidelity polymerase and see if the situation improves. You could also try doing fewer cycles, or drop your extension temp a bit, or try another E. coli recipient strain...

But, a further question -- why do you need more than one mistake-free clone?



I don't. I was afraid I wouldn't be clear in describing my problems, and apparently I wasn't. If I get one mistake-free clone, I'm happy. But I'm doing 6 different constructs. Four of them are gene fusions to FLAG and the other two are mutagenesis projects. So when I'm talking about the different clones, it's the 6 different constructs (4 were gel purified for cloning, 2 were PCR purified) and so forth.


I've contemplated using another polymerase. We used to use Pfu Ultra II, and that always worked well, but then I saw Phusion one day, and it was about 1/2 the price for 2x the reactions. We tried it out, and it seemed to work great. We had been using it for a couple years before we (I) ran into the current issues. I don't think it was a bad stock of the polymerase, because we go through it pretty quickly, and I'm sure we've ordered new a few times since the first problem came up. I will try doing the current "bad" construct one more time with Phusion and if it gives poor results I will try Pfu Ultra II again.


As for the E. coli strain, I'm currently in a back and forth with Stratagene regarding the XL1 Blue cells to see if they have any ideas. I'm guessing they'll tell me that our freezer stock may just be old and to get a new one. I'm not sure when the freezer stock was put in there, if it's from an original or a subculture along the way, but I think we got that strain with the plasmid, and the plasmid has been in the freezer since 1999. Who knows.


Anyway, I appreciate all the thoughts and considerations. Maybe something will work, or I'll just get lucky and find a clone out of a few isolates without a mutation and just go from there.

#10 fishdoc

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Posted 27 August 2009 - 01:32 PM

OK, so here's where I am with this problem.


I have been in contact with NEB regarding their Phusion polymerase, and they've basically given me the "I have no friggin' clue" response. The DMSO I had been using was not a problem (according to them), my reaction conditions are fine, they had no history of complaints about that lot of Phusion, etc. They suggested maybe the dNTPs were out of whack, and asked if we made our own or bought commercially. We purchase from Bioline, and aliquot out to avoid to much freeze-thawing, and I've been through multiple aliquots with the same results (maybe the original stock from Bioline is bad?)

I then found some polymerase from about a year ago (Pfu Ultra II from Stratagene) and decided to use that side by side with the Phusion. Both amplified well, and I inserted a portion of each PCR product into pBluescript and screened with X-gal. For the Phusion, I got 15 white colonies and for Ultra II, only 5. I did colony PCR on each, and 3 of the 5 Ultra II clones had the correct sized product while only 1 of the 15 Phusion had the correct size. I sent the 4 good ones for sequencing. Of the 3 Ultra II clones, 2 of them had a single point mutation in them, but the third was fine. The Phusion sequence was very odd. The colony PCR using T3/T7 primers showed an insert, but the sequencing showed no insert. At this point, I really didn't care, because I had the one good one from Ultra II.

In the meantime, I was also constructing some complementation plasmids, and decided to use Ultra II. Today, I got back the sequencing for 2 of them, and in both cases, only 1 of 3 clones was correct sequence. Each of the others had 1 point mutation, all in different positions.

Thus, it seems it is not the polymerase that is causing these point mutations, because I am getting them with two (supposed) high fidelity enzymes.

Aside from one particular gene I'm trying to clone, I've ultimately gotten 1 good clone to use for further manipulation, but this level of mutation is very troubling.

The common reagents used in both the Phusion and Ultra II reactions are the dNTPs, the primers, and the template. The template is genomic DNA stored at 4 C, and it's been in there a couple years. The only other thing I could think of is that the 35 cycles is too much, thus causing both the Ultra II and Phusion to lose some fidelity. However, I'd guess you'd still expect a good majority of your amplicons to be of the correct sequence if that were the case.

I'm really at a loss for ideas on what could be causing this. We've got new Phusion and new Ultra II on the way, and I'm going to do some more side-by-side stuff to test them.

Any thoughts or suggestions?

#11 Warren

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Posted 30 August 2009 - 02:27 PM

In the meantime, I was also constructing some complementation plasmids, and decided to use Ultra II. Today, I got back the sequencing for 2 of them, and in both cases, only 1 of 3 clones was correct sequence. Each of the others had 1 point mutation, all in different positions.

Any thoughts or suggestions?


My first guess when reading this thread was UV light....but I wonder...you say you "got back the sequence"....did you look at the actual chromatograms in the area you see a mutation, or are you assuming the sequencing is correct? Computers can make errors in base calling and by looking at the chromatograms you can additionally get a feel for how well the sequencing reactions worked, that could also be an indicator of the quality of the template. One thing I would try and be certain of is that the sequence you are getting is the actual sequence -- if not all the other experiments are null.

Another thing you might try, since you are going to great lengths to solve this problem, is do the "opposite" experiment and try Taq and see what the error rate with it is compared to your high fidelity enzymes. There are also alot of tricks you can do to increase "apparent" fidelity, like reduce the cycles (as others have suggested) or start with more template. You could even use one of your "correct" clones as a template, for experimental purposes.

I'd be interested to hear how things turn out...Warren..

#12 fishdoc

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Posted 30 August 2009 - 02:47 PM

In the meantime, I was also constructing some complementation plasmids, and decided to use Ultra II. Today, I got back the sequencing for 2 of them, and in both cases, only 1 of 3 clones was correct sequence. Each of the others had 1 point mutation, all in different positions.

Any thoughts or suggestions?


My first guess when reading this thread was UV light....but I wonder...you say you "got back the sequence"....did you look at the actual chromatograms in the area you see a mutation, or are you assuming the sequencing is correct? Computers can make errors in base calling and by looking at the chromatograms you can additionally get a feel for how well the sequencing reactions worked, that could also be an indicator of the quality of the template. One thing I would try and be certain of is that the sequence you are getting is the actual sequence -- if not all the other experiments are null.

Another thing you might try, since you are going to great lengths to solve this problem, is do the "opposite" experiment and try Taq and see what the error rate with it is compared to your high fidelity enzymes. There are also alot of tricks you can do to increase "apparent" fidelity, like reduce the cycles (as others have suggested) or start with more template. You could even use one of your "correct" clones as a template, for experimental purposes.

I'd be interested to hear how things turn out...Warren..



I have looked at the chromatograms, and in every instance save one, the computer made the correct call, based on the peak anyway. The one time it was incorrect was when the person that does all the sequencing for us looked at the chromatogram and called a T instead of a G. The chromatogram showed a small T peak, and the following base was a G. That G actually had a little shoulder before it that was above the peak for the T, so I assumed it was a miscalled G. However, another possibility is that the template to be sequenced was a pretty even mix of G and T at that position, and that's why there was a small T peak and a G shoulder. I'm not familiar enough with sequencing to know if that is a valid thing to happen. As for the sequence of the original, it's all from the genome sequencing project of our bacterium. I think the entire genome is at least 8 fold coverage (maybe more), so I think the template (sequence) I'm basing it on should be correct.

As for UV, many of these constructs are not being gel purified, but rather purified directly from the PCR reaction, so there is no UV exposure.

As for "going to great lengths", I was to begin with, but it began eating up a lot of time and resources to do side-by-side reactions and sequencing, and everything else. While I'm not happy at all with the poor sequences I've been getting, I can work through it as long as I'm getting some correct that I can carry on to the next step, at least in the short term. If this becomes a chronic problem, then I (we) will delve into it further. Because there has been relatively little response to this problem, and because a few google searches here and there have revealed no other similar issues with high fidelity enzymes, I'm assuming it's something on our end. Either a bad batch of polymerase, someone leaving it sit out too long, or perhaps the -20 isn't actually keeping at -20 (we are in a pretty humid climate, so the -20 frosts up pretty quickly and doesn't seem to work efficiently if it gets too frosty).

I was talking to my PI the other day about it, and for every reason we think of that it could be happening, we can think of 30 other reasons those aren't logical. But since we ARE having the problems, one of those seemingly illogical explanations really is quite logical, we just don't know it.

In any event, I will keep updating this thread as I find out any more information (if I find out any more information). We received new Phusion and new Pfu Ultra II on Friday, so this week I will be doing some more reactions and cloning, so maybe I will have some info on the new enzymes sometime next week.

#13 phage434

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Posted 30 August 2009 - 04:13 PM

I would immediately try new dNTP solution, or use a master-mix. dNTPs can easily go bad, and could explain your poor PCR performance. Also, how certain are you that the "errors" you are seeing are not real mutations in the template sequence?

#14 fishdoc

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Posted 30 August 2009 - 05:12 PM

I would immediately try new dNTP solution, or use a master-mix. dNTPs can easily go bad, and could explain your poor PCR performance. Also, how certain are you that the "errors" you are seeing are not real mutations in the template sequence?



Yes, new dNTPs are on order, too. We usually order a few tubes and aliquot as needed to lessen freeze-thaws. I had used different tubes from the freezer, but perhaps they weren't too good.

I don't think there errors are in the templates sequence, because they're in different places in different clones of a single construct. If the sequenced clones had the same "mutations" in the same place, I'd consider that my sequence of the template was just wrong, but that hasn't been the case. Furthermore, some of clones do have the expected sequence (no mutations) when cloned, but if I sequence multiple clones, I've had 2/3 have the mutations (different mutations in the two), but the other is good. In one case, I had 5 clones of one construct. Each one had between 3 and 5 mutations, and each mutation was in a different position. The incorrect bases haven't been consistent, which is why I don't believe they're the actual sequence.

#15 Warren

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Posted 30 August 2009 - 05:50 PM

I would immediately try new dNTP solution, or use a master-mix. dNTPs can easily go bad, and could explain your poor PCR performance. Also, how certain are you that the "errors" you are seeing are not real mutations in the template sequence?



Yes, new dNTPs are on order, too. We usually order a few tubes and aliquot as needed to lessen freeze-thaws. I had used different tubes from the freezer, but perhaps they weren't too good.



I think bad dNTPs are FAR more likely than two bad batches of thermostable polymerases that just happen to affect fidelity. Those polymerases are TOUGH (try to heat inactivate one :( and I'd say if your freezer wasn't holding temp you'd be far more likely to notice a problem in a more sensitive enzyme (like T4 DNA ligase) but I doubt that is the problem. I'd bet dollars to donuts that if its a component of the reaction, its the dNTPs (plus your looking for something specifically affecting fidelity). Although it seems like a waste of time and money if you figure it out you will save alot of time and money in the future! It costs a lot of money to buy those high fidelity enzymes, then do all that sequencing to find a correct clone because of the errors -- might as well use Taq! Good luck..Warren..




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