pmc-at - PCR Kinetics

6/4/13
Minutes of telecon 6/4/13 PM (including email exchange leading up to it)

KM: I could understand the Fluorescence data and it says that in trial 1, we could achieve 96% (I have calculated this number based on the initial and final concentration) efficiency in 48 seconds. Whereas in trial 2, the same efficiency was obtained in 40 seconds. This may be probably due to the early melting.

SM:

1. I would say that the two trials are quite similar.
2. I had incorporated an initial step in the program to measure the fluorescent signal of the ds DNA at the start of the expt. The starting RFU was ~10000. However the melt kinetic curve starts at ~4000. At this point I don’t know whether there was some instantaneous melting (to drop signal from 10000 to 4000 or this is simply a limitation of the PCR software that there is a lack of continuity between cycling steps.

KM: Does the absorbance data also signify the same conclusion?

SM:

1. In the UV expt the melting appears slower (it takes ~5-7mins for the curve to plateau as opposed to ~40secs in the PCR machine).
2. The curves obtained in the UV expt need smoothing/ fitting.
3. At this point I do not know why the Evagreen and Uv methods give different rates of melting. It has to be noted tho’ that the DNA conc in the UV expt is lower and the total volume of sample is higher.

KM: Also, the initial concentration of the DNA is out of the range of typical PCR conditions. I would suggest to use 5nM to 0.5 micro Molar concentration and calculate the melting time. Because, this is the range of the DNA that we encounter in a typical PCR. So we may want to consider to do these experiments.

SM: At this point I cannot devote more time for these expts. My priority is to finish the extension kinetics work.

On the extension experiments:

We should also measure the rate in 45, 55 and 65 deg C.

SM: As per Dr Chakrabarti’s advice, I will assay at 3 temps (50, 60 and 70degC). If reqd, we will then fill the gaps and assay at 3 more temp.s

We have agreed on the following experimental parameters:
Template conc: 200nM
Enzyme conc: 0.36nM
Equilibration : yes: 30mins at assay temp
dNTP conc range: 2, 20, 100, 200, 1000, 2000 uM.
Assay temps: 50, 60, 70oC (initially)

From: Karthikeyan Marimuthu [mailto:[email protected]]

Sent: Tuesday, June 04, 2013 9:44 AM

To: Sudha Moorthy

Subject: RE: extension kinetics

Hi Sudha,

I have understood the following on the Melting experiments results.

There are two different data that are based on Fluorescence and absorbance.

I could understand the Fluorescence data and it says that in trial 1, we could achieve 96% (I have calculated this number based on the initial and final concentration) efficiency in 48 seconds. Whereas in trial 2, the same efficiency was obtained in 40 seconds. This may be probably due to the early melting.

Does the absorbance data also signify the same conclusion?

Also, the initial concentration of the DNA is out of the range of typical PCR conditions. I would suggest to use 5nM to 0.5 micro Molar concentration and calculate the melting time. Because, this is the range of the DNA that we encounter in a typical PCR. So we may want to consider to do these experiments.

On the extension experiments:

We should also measure the rate in 45, 55 and 65 deg C.

I am ok with the second set of concentration. But instead of 1, 2 and 20 micro Molar, we may use 1, 10 and 50 micro Molar.

Karthik.

From: Sudha Moorthy [mailto:[email protected]]

Sent: Monday, June 03, 2013 12:10 PM

To: Karthikeyan Marimuthu

Subject: Re: extension kinetics

Importance: High

Karthik,

Hope you had a chance to take a look at the Lambda DNA melting kinetics results I had posted on Friday.

I am waiting for template DNA (which should arrive by Wednesday) to start the extension kinetics expts., To refresh here are the test conditions:
Template conc: 200nM
Enzyme conc: 0.36nM
Equilibration : yes: 30mins at assay temp
Test Assay temps: 50, 60, 70oC

Would like to confirm with you the dNTP concentrations we are going to use:
I had originally suggested (for purposes of starting the discussion) the following: 20, 50, 100, 200, 500, 1000 uM

However, since you advised that 20uM itself might be excess, I am wondering if one of the following ranges might give us more information:

1. 2, 20, 100, 200, 1000, 2000 uM.
2. 0.2, 1, 2, 20, 200, 2000 uM

It would be good if you can get back to me regarding this by tomorrow afternoon. In fact if you have time, maybe we can talk tomorrow afternoon or Wednesday morning so that we are agreed on the experimental parameters. Please suggest a convenient time.

Regards,

Sudha.

5/31/13
Karthik,

Please find attached a power point file with results of melt kinetic experiments using phage lambda DNA.

Since we do not have any DNA of ~500bp and it is too expensive to synthesise or purchase, Dr Chakrabarti advised to perform an informal experiment with Lambda DNA to see if we can get any meaningful data.

The experiment is as follows: Double stranded phage Lambda DNA (~45kb) in 1X Taq Polymerase buffer (with 2mM MgCl2) was incubated at 95oC and the amount of ds DNA was measured as a function of time over a period of 25-45mins. The experiment was tried in the PCR machine (Evagreen dye was added to the reaction mix to a final conc of 1X) as well as in the UV-Vis spectrophotometer (Absorbance (260nm) was measured without any dye). A buffer blank sample (that contained no DNA) was simultaneously run in both cases.

For the experiment in the UV-Vis spectrophotometer, the kinetic application was used. Briefly, this application allows the user to set the temperature for measurement and plots a curve of Abs vs time. The length of incubation is user determined. In a kinetic run, the software prompts for the addition of the active agent (in this case, the DNA). Measurements are started at the end of a 2min timer or when the user hits OK.

The UV expt can thus be performed in 2 ways (the difference between the two approaches is tabulated below):
Test 1:

1. Prepare the reaction samples for buffer blank and lambda DNA but do not add DNA.
2. Transfer the reaction sample to cuvettes. Start the run.
3. Once the instrument reaches 95oC, it prompts for addition of DNA.
4. Now, add the DNA to the DNA cuvette, hit OK and allow for measurements to start.

Test 2:

1. Prepare the reaction samples for buffer blank and lambda DNA, this time add the DNA and mix thoroughly.
2. Transfer the buffer blank sample to the cuvette. Start the run.
3. Once the instrument reaches 95oC, it prompts for addition of DNA.
4. Now insert the DNA cuvette, hit OK and allow for measurements to start.

Test 1	Test 2
The buffer for the DNA is already at 95oC when DNA is added. Thus the DNA will reach 95oC in a very short time. So we can probably presume that all the melting of the DNA is taking place at 95oC only, as required.	The DNA in its buffer is at RT. When the cuvette is inserted into the instrument, the DNA takes a longer time to reach 95oC from RT, during which time some melting may be taking place.

Hope this experiment will give you the information you want. I have other projects currently running that need attention and will not be able to spend more time on this work at this point.
Let me know if you have any questions or if you want the raw data (I can send that in Excel).
Sudha.
Lambda Melt Kinetics May 2013

5/23/13 Minutes of telecon 5/22/13 (please see emails 5/21 to 5/23 for background)

KM: If you feel that the melting of 45kb base pairs will be complete (without forming secondary stages) in a specified time, then, we can use this DNA. In fact this will be the most appropriate for our analysis. We would like to find the lower bound for the melting rate constant that corresponds to relatively long DNA.

SM: Lambda DNA is very big (~45kb), and not suitable for a first time study for the following reasons:

1. The one time that I have performed melting studies on Lambda (very slow ramp down from 95 to 25degC), it showed domain melting.
2. We have no idea how the long DNA (~500bp) is going to work, nor do we know how the program will behave with the long DNA,
3. Literature for Lambda DNA melt studies is very dated.

I have to see if PCR products of the required length can be generated in the lab or purchased. Also whether it is viable to synthesize 500bp long DNA molecules. At least 2-3 will be required to validate the simulation.

SM: The extension kinetics work is still not complete, provided nothing goes wrong, this will still take 8-10 weeks. During this time it will be difficult to start a new set of experiments particularly if these need standardization.

Given that we will be using new DNA molecules, there will be some amount of standardizations involved (trial runs, dye concentration, instrument parameters, choice of UV-Vis vs fluorescence etc)

While the Taq polymerase assays are going on, the materials can be obtained and the protocol established to the extent that it can be done.

4/16/2013,
Hi Sudha,Please find my comments in red color.Thanks,Karthik.
4/15/2013

Karthik,

Please see my responses to your two posts dated 4/15/13
Melting Experiment:

Hi Sudha,

I think I have already wrote about Melting kinetics. We need to also determine the melting kinetics parameter.

Protocol 1:At 90 deg C, (We need to somehow ensure that from room temperature, reaction mixture is heated to 90 or 95 deg C very quickly), just measure the time required for the complete melting. We don't need the time dependent Fluorescence signal and we just need the time required for the complete melting at 90 deg C. From this information, it is possible to determine the rate constant for the melting reaction.
Are you particular that the temp. should be 90degC? Why? The oligomer pair I have used so far has a GC content of 50% and is completely melted even at ~85degC (probably lower). I also have oligo pairs that do NOT melt even at 90degC. The ramp rate from RT to 90degC (or whatever temp you choose) can be set at the highest value ensuring that the mixture is heated as quickly as possible. To measure the time required for complete melting, we would still have to measure the fluorescence/ absorbance signal (since only by following the curve will we know that the ds DNA has completely melted).
KM: We need to measure the time for complete melting at 90 deg C for different sequences. Because, if we know that most of the sequences ( not the ones that make the secondary structure) melt at 90 deg C, then, using an average time (average will be calculated based on many sequence data) that takes for the complete melting, it is possible to derive an universal rate constant for the melting process. At very high temperature since the forward rate constant of melting process is too high compared to the reverse rate constant, this reaction can be assumed to be irreversible. Also, if the time taken for complete melting of different sequences are within a specific range, we can make appropriate approximations in estimating the melting rate constant. That is why we need to know the time required for complete melting for various sequences at 90 deg C or 95 deg C.
Protocol 2:As we measured the time dependent Fluorescence or absorbance signal to measure the double stranded DNA concentration, if it is possible to measure the double stranded DNA concentration from time t=0 (in this case Fluorescence signal will decrease from t=0 and reach a steady state value) in a certain time interval, we can fit this data and determine melting kinetics parameters.
If I understand correctly, the temp has to be increased from RT to 90degCand the fluorescent signal measured during the increase of temp and the plotted against TIME, is that correct? With the software we have the melting curve cannot be plotted against time, it is only plotted against temp. Also, this experiment describes the complete melt run that I have already done, only I decreased the temp from 90 to 25degC.
KM: Ramp from RT to 90 deg C and fix the temperature to be 90 deg C and measure the time dependent fluorescent data. I don't know whether the given DNA will melt completely during the ramp stage. If it does, then we will have to think another way of doing this.
The melting kinetics parameter is more important than annealing reaction parameter. So, please prioritize this experiment (I hope you don't need to do much change in the current annealing protocol).
The priority for me right now is the polymerase activity assays to measure extension rates. I can get back to these experiments after the activity assays are done.KM: OK. If possible and if you find sometime in between, I request you to try this experiments. But as I you said, the priority is on the polymerase activity assays.
Thanks,

Karthik.

4/15/2013
Hi Sudha,
I have gone through the slides.

As you have mentioned, the sensitivity of Fluorescence spectroscopy is better than the absorbance. Based on this data, I can start doing some quantitative check.

1) Both Fluorescence and absorbance signals reaches steady state after some time. Also we know the Fluorescence and absorbance value at time t = 0. Essentially we know initial and final concentration of the double stranded DNA molecules.

Although we know the fluorescence and absorbance values at time t = 0, at this point I cannot convert these values to the amount of ds DNA (see arguments in the power ppt presentation). What we do know for sure are the following:
a) The initial concentration at which the ss oligos were added to the reaction mix.
b) If the protocol starts at 90degC, we all that all the DNA is completely melted and in ss form, therefore the concentration of ss DNA is the same as initial conc., and there is no ds DNA.
c) In the absorbance spectroscopy expt., the absorbance value at the hold temp is a combination of ds and ss DNAs (calculation at time t = 0 of the kinetic run of the ds DNA present; I need to think about how to get the conc of ds and ss DNA from this.)KM: Is it not possible to use a linaer approximation to calculate the amount of single and double strands. Total Absorbance = x*(absorbance at 90) + (1-x)*absorbance at 25. From this equation calculate x and x is the single stranded DNA and 1-x is the double stranded DNA.
d) Since the fluorescent signal is a direct measure of the amt of ds DNA, the calculation here should be easier, however under the conditions used to get good resolution curves for the temp.s at which the kinetic run was performed, the fluorescent signal was already saturated at 65degC. We will need standardization of the experimental conditions to get equally good kinetic curves for both 70deg as well as 25deg and then calculate the initial conc.KM:The Fluorescent signal is saturated may be because at 65 deg C, 100% double stranded DNA formed or all ssDNA is converted in dsDNA. (I can check this theoretically for a given sequence). So if we can choose a sequence that is not stable even at lower temperature ( typically small sequences with less GC content) we may probably conduct the experiment till 30 or 25 deg C.
2) In order to check whether the reaction reached the equilibrium, we can do a theoretical analysis and compare the theoretically obtained DNA concentration and the concentration based on the Fluorescence and absorbance values. This will suggest the accuracy of our experimental protocol.

3) So, please provide me the calibration curve or convert the Fluorescence and absorbance values in to a double stranded DNA concentration. I will do the calculation and give me comments.
I will work on this, however the priority for me right now is the polymerase activity assays to measure extension rates.KM: Ok.
4) Since the sequence has more GC content, the Fluorescence signal saturates at 60 deg C.
I am not sure I understand this. The oligomer pair I have used has a GC content of 50%.KM: Please refer my comment under point d.
In order to check the kinetics at lower temperatures, it would be easy if we can choose short and also low GC content oligomers. Still, for this sequence, the selected window of temperature looks promising and it will give a good idea about the kinetics of DNA annealing reaction.
5) In order for me to do some theoretical calculations, I need the following data1) Salt concentration ( You have mentioned the Mgcl2 values but I would like to know whether the buffer has Na or K salt.2) Initial concentration of the single strand molecules ( I hope it is 0.5 micro molar).
Initial conc of each oligo is 0.5uM in the fluorescence expt.
It is 1uM in the absorbace expt.
The buffer contains 50mM KCl. I also add 2mM MgCl2.
What do you feel about the choice between Fluorescence and absorbance spectroscopy?
I have to see how easy/ difficult it is to get the initial conc at time t= 0, then we’ll see. Right now neither prcl is giving us what we want.

4/15/2013
Melting Experiment:
Hi Sudha,
I think I have already wrote about Melting kinetics. We need to also determine the melting kinetics parameter.
Protocol 1:At 90 deg C, (We need to somehow ensure that from room temperature, reaction mixture is heated to 90 or 95 deg C very quickly), just measure the time required for the complete melting. We don't need the time dependent Fluorescence signal and we just need the time required for the complete melting at 90 deg C. From this information, it is possible to determine the rate constant for the melting reaction.
Protocol 2:As we measured the time dependent Fluorescence or absorbance signal to measure the double stranded DNA concentration, if it is possible to measure the double stranded DNA concentration from time t=0 (in this case Fluorescence signal will decrease from t=0 and reach a steady state value) in a certain time interval, we can fit this data and determine melting kinetics parameters.
The melting kinetics parameter is more important than annealing reaction parameter. So, please prioritize this experiment (I hope you don't need to do much change in the current annealing protocol).

Thanks,
Karthik.
4/15/2013
Hi Sudha,
I have gone through the slides.
As you have mentioned, the sensitivity of Fluorescence spectroscopy is better than the absorbance. Based on this data, I can start doing some quantitative check.
1) Both Fluorescence and absorbance signals reaches steady state after some time. Also we know the Fluorescence and absorbance value at time t = 0. Essentially we know initial and final concentration of the double stranded DNA molecules.
2) In order to check whether the reaction reached the equilibrium, we can do a theoretical analysis and compare the theoretically obtained DNA concentration and the concentration based on the Fluorescence and absorbance values. This will suggest the accuracy of our experimental protocol.
3) So, please provide me the calibration curve or convert the Fluorescence and absorbance values in to a double stranded DNA concentration. I will do the calculation and give me comments.
4) Since the sequence has more GC content, the Fluorescence signal saturates at 60 deg C. In order to check the kinetics at lower temperatures, it would be easy if we can choose short and also low GC content oligomers. Still, for this sequence, the selected window of temperature looks promising and it will give a good idea about the kinetics of DNA annealing reaction.
5) In order for me to do some theoretical calculations, I need the following data1) Salt concentration ( You have mentioned the Mgcl2 values but I would like to know whether the buffer has Na or K salt.2) Initial concentration of the single strand molecules ( I hope it is 0.5 micro molar).
What do you feel about the choice between Fluorescence and absorbance spectroscopy?
4/5/13

Karthik,
Am attaching 2 power point files that summarise the annealing/ melting kinetics expts (to date) run with the new Agilent program on the UV-Vis spectrophotometer and the Fluorescence spectrophotometer.
The protocol followed for both is the same (start at 90deg>> ramp down to desired hold temp>> kinetic run at hold temp till equilibration).
1. The hold temperatures were chosen based on the Tm measured under identical conditions.
2. Limitations/ advantages/ differences of one over the other are listed in the Fluorescence spectroscopy file.
3. These files simply summarise the data. I can pass on actual data points if you require them.
4. Calculations:
a. UV Absorbance spectroscopy: As we have discussed before, both ds and ss DNA is quantitated at A260. The absorbance measured during the thermal and kinetic run represents a combination of ds and ss DNA. We know the following facts:
i. Concentration of each oligo in the reaction mix.
ii. At 90oC, there is no ds DNA, therefore the A260 value represents ssDNA only
iii. Similarly, at 25oC, there is no ssDNA, therefore the A260 value represents dsDNA only
Can we use these facts to calculate the concentration of ds DNA from the A260 values of the thermal/ kinetic run?
b. Fluorescence spectroscopy: Since Evagreen is a ds DNA specific dye, the fluorescent signal directly represents the amount of ds DNA present in the system. However, to calculate the conc, either a calibration curve would be required or a correlation with the signal of the reaction sample when all the DNA is in the ds form i.e., at 25oC. Since our measurements are made at different temperatures, I felt the second approach i.e., correlation with the signal of the reaction sample at 25oC would be better. However, instrument parameters that were used to perform the thermal/kinetic run resulted in signal saturation at 25oC. Thus to make meaningful calculations, more standardization will be necessary. However, the attached file shows that the method works.
Please go through the data and we can discuss as per your convenience.
Sudha.
Fluorescent spectroscopy
UV-Absorbance Spectroscopy

3/26/13
Karthik,
Three excel files uploaded on 3/20/13. The files can be accessed from "Pages and Files".
Sudha.
03.25.13Hi Sudha,Sorry for the late response. There is no attached file here. (is it the one that you have attached before and updated the same).I have created a separate page for the discussion on the extension kinetics paper. Please see that page for our recent discussion on your paper.I will include more material in this page tonight.Karthik.03/20/13
Karthik,

As mentioned yesterday, I have made a new program with protocol 1 (start at 90>>ramp down to 68/ 74deg>> kinetic run at end temp). Am attaching the following excel files
1. 74deg expt
2. 68deg expt
3. Comparison of 68-74deg expt (here the graphs are plotted in excel)

My feeling from these expts is that the amount of oligos taken for each melt run needs to be increased to be able to see a significant absorbance change during the kinetic run. Right now (with 0.5uM oligo), the variation is very slight (could be noise due to peltier equilibration).

However, the program seems to run and am planning to run higher oligo conc.s tomorrow.
Sudha

03/19/13

Karthik,

Please see my responses below:
1) The absorbance is directly proportional to the amount of single strands. In other words, higher the absorbance value indicates the high concentration of the single strands and lower value of the absorbance indicates the more double strands. If this is the case, the absorbance and 74 deg C will be greater than that at 65 deg C. But, as per the saturated the absorbance values, absorbance at 65 deg C is more that of at 74 deg C. Please correct me if I am wrong in my understanding on the absorbance.

Both dsDNA and ssDNA absorbance is measured at 260nm (as we have discussed earlier). In a mix of ds and ss DNA, as during a melt run, yes the absorbance is directly proportional to the concentration of ssDNA: as the amt. of ssDNA increases due to melting, the absorbance also increases.
To compare the 68 and 74deg data, we need to compare the starting absorbance at 25deg. Also the change in absorbance during ramp up from 25 to 68/ 74deg. These no.s are as follows:

Expt	Start Abs (25deg)	Calculated conc of dsDNA (ng/ul)	Calculated conc of dsDNA (uM)	End Abs	End Abs-Start Abs
68deg expt	0.3940551	19.702753	0.74uM	0.4065934	0.0125383
74deg expt	0.3654431	18.272154	0.7uM	0.3994165	0.0339734

2) Once we can correlate this data with the concentration of single strand or double strand, we can fit this data based on the annealing kinetics that I explained previously.

If we have absorbance at 90deg (at which temp, this oligo pair is completely melted), the total amt of ssDNA can be cal’d (similar to the calcn above for ds DNA). However, at 68/ 74deg all the ds DNA is not melted, so I am not sure how the end absorbance can be correlated to DNA conc.

3) The initial increase in absorbance for 10-20 seconds, could be due to temperature deviation. Is this correct?

However close the measurements in the kinetic run are, the absorbance in the second/ third observation goes up. (please note we saw something similar even in the expts. conductec on the PCR machine). This appears to be a limitation of software/peltier temp control. This “hump” might disappear if we equilibrate the sample for a few mins, however we run the risk of “missing all the action” from the first few secs.

I am trying some tests to see if we can work around it, let me see how these go.

Sudha.

03/18/13Hi Sudha,

Thanks for the data.
I have gone through the data quickly and have few questions.
1) The absorbance is directly proportional to the amount of single strands. In other words, higher the absorbance value indicates the high concentration of the single strands and lower value of the absorbance indicates the more double strands. If this is the case, the absorbance and 74 deg C will be greater than that at 65 deg C. But, as per the saturated the absorbance values, absorbance at 65 deg C is more that of at 74 deg C. Please correct me if I am wrong in my understanding on the absorbance.
2) Once we can correlate this data with the concentration of single strand or double strand, we can fit this data based on the annealing kinetics that I explained previously.
3) The initial increase in absorbance for 10-20 seconds, could be due to temperature deviation. Is this correct? If this is the case, can we normalize this data in such a way that we will account from the point at which the concentration values start decreasing.
Thanks,
Karthik.
03/18/130314 0.5uM 68deg 1.xlsx
Karthik,

Am attaching two excel files with preliminary data from the UV absorbance annealing experiments.

1. Since I havent yet resolved issues with Protocol 1, these expts are both protocol 2 (start at 25>>ramp up to ~70>>kinetic run at end temp).

2. Each excel file has 2 sheets, one with the expt parameters and one with the data. The actual data points are available. I have also copy-pasted the graphs from the spectrophotometer data.

3. Two formats of the kinetic run graphs at pasted: same scale as the thermal run, and a zoom up so that the pattern in the first 1min or so is clear.

Please go through this data, feel free to call me if you want and we can discuss it (in fact it might be good to plan for a telecon)

0318 0.5uM 74deg.xlsx
Sudha.

03/15/2013
Hi Sudha,

Thanks for the explanation. I have some idea to correlate the absorbance of mixed state ( I mean mixture of both double strand and single strands).

1. We know the correlation between the double stranded and absorbance and Single stranded dna and absorbance. Let us assume at the mixed state, x fraction of double stranded DNA got melted in to single stranded DNA and we know the absorbance of the this state as well. Now following equation can be used to find the amount of single strands or double strands.

X* absorbance of double stranded DNA (this one was measured at lower temperature) + (1-X)* absorbance of single stranded DNA molecule (this one was measured at higher temperature) = absorbance at any stage.

Here X can be a mole fraction or weight fraction.

Essentially we know mass balance between the ds DNA and ss DNA and if make use of it, we should be able to derive the above formula. Please let me know whether this is correct.

2. I don’t have any specific preference over one protocol. Whichever is convenient and that can give a good data, then we can follow that.

I will post some notes on the extension reaction paper in the wiki tomorrow.

Thanks and Regards,

Karthik.

03/14/13
Karthik,

Please see my responses below:

Hi Sudha,

I and Raj have discussed about the extension kinetics results that were presented in the paper. Both of us have thought that using the data that were presented in the paper, it is possible to derive the extension rate parameter. I have raised few questions on estimating the Michaelis Meten kinetics parameters and gave my explanation on why should we plot the data differently. Raj will go through my explanations and get back to us on estimating the Michaellis Menten kinetics parameters. He will also comment on the possible experiments and simulations that can be performed using PCR machine to validate kinetic parameters. This experiments and simulations will also give some idea about the robustness of the estimated kinetic parameters.

In addition to this extension reaction part we need to do experiments on the Melting and Enzyme binding.

Our PCR simulation studies revealed that enzyme binding reaction is the rate limiting and hence, this will affect the overall kinetics of the PCR. The current enzyme binding kinetic parameters that we have used in our simulation is based on equilibrium thermodynamics data (Datta and Licati) of Taq enzyme binding reaction and our own assumptions. In order to validate our assumptions, we need to experimentally find the forward and reverse rate constant of enzyme binding reactions. I have given the above reference, Datta and Licati, which discuss the experimental procedure to determine the equilibrium constant of an enzyme binding reaction, based on Fluorescence anisotropy assay method. Can you go through the experimental section of the above paper and determine whether it is possible to conduct those experiments in PMC? Also, it would be nice if you can comment on conducting kinetics experiments using the above assay.

I will read the paper and respond to this in a separate mail.

We have done the annealing study of the small primer molecules and I believe we are waiting for the absorbance spectroscopy method. Once we receive an update on the absorbance spectroscopy method, we can continue to work on this.

I am working on smoothing out and tweaking the program for the absorbance spectroscopy to get good, clean curves. Basically what the program does is start a thermal run at the start temp (user input), end thermal run at the end temp (user input) and immediately start a kinetic run at the end temp of the thermal run (length of kinetic run is also user determined). However, we still essentially have two kinds of protocols:

Start at the high temp (say 90 or 95deg), ramp down to desired temp (say 70deg) and do a kinetic run at 70deg. Here we start with completely melted DNA and measure the rate of annealing at the desired end temp. The variations I am planning are as follows:
1. Start temp 90deg>> ramp down to 80deg>> kinetic run at 80deg
2. Start temp 90deg>> ramp down to 75deg>> kinetic run at 75deg
3. Start temp 90deg>> ramp down to 70deg>> kinetic run at 70deg
4. Start temp 90deg>> ramp down to 68deg>> kinetic run at 68deg
5. Start temp 90deg>> ramp down to 65deg>> kinetic run at 65deg

Start at a low temp (say 45 or 50deg), ramp up to desired end temp (say 70deg) and do a kinetic run at 70deg. Here we start with annealed DNA and measure the rate of melting at the desired end temp. Here again, the same end temps as above will be used but the start temp for each run would be 50deg.

All these runs will be completed with the same oligo pair that I used for the expts on the PCR machine (40mers; 50% GC); whose Tm information I have.

Also, I have to standardize parameters like ramp rate, signal averaging time, data collection interval etc before I do the runs.

Finally, if you would need data with oligos of different GC content, those will be additional expts.

It appears that protocol 1 is more difficult to accomplish even on the spectrophotometer (though I am currently working on methods to get around that); but do you prefer one protocol over the other? Protocol 2 works more easily (it may be noted that this was true even for the expts. performed on the PCR machine).

Finally, we will have to do some study to understand the kinetics of melting of long DNA molecules. Is it possible to determine the melting time for different length of DNA molecules (length more than 500 bp)? For example, we will consider a double stranded DNA and melt that for different times. Each time we will measure how much of double stranded DNA is converted in to single stranded DNA. Even if we can have only the time required for complete melting, that is sufficient. We will need this data at 90 deg or 95 deg C alone. At this temperature, we will assume that melting is an irreversible reaction with first order kinetics. So, if you can provide me the time required for the complete conversion of double strand molecules in to single strand, I can determine the rate constant. Note that here we will have to consider DNA with more than 500 base pairs. Please let me know whether it is possible to do all these experiments.

For a DNA molecule >500bp, we can presume that the Tm > 95deg. In that case, the expt you have suggested is not different from what I am currently doing with the 40mer oligos. So using Protocol 2 from above, we would heat the DNA up from 25deg to 95deg, and do a kinetic run at 95deg which would measure melting at 95deg. Please note that it is likely that the DNA would be partially melted when we start the kinetic run (during the ramp up from 25-95deg which can also be measured).

The issue that I have all this absorbance protocols is as follows: We measure the absorbance of both ssDNA as well as dsDNA at 260nm. For the 40mer oligos, during a melt run from 25 to 95deg absorbance increases as the amount of ssDNA increases. At 25deg (all DNA is annealed and therefore ds) we can correlate the absorbance to the (known) conc of dsDNA. Similarly at 95deg (all DNA is melted and therefore ss) we can correlate the absorbance to the (known) conc of ssDNA. However, I am unclear how we will determine the conc of ds DNA at the end of thermal run-start of kinetic run junction since we will have some dsDNA and some ssDNA in the mix at that point. Also depending on the temp at which we want to do the kinetic run, this proportion of dsDNA to ssDNA will change.

This will be a non-issue if we do fluorescence spectroscopy (say using SYBR) since the signal is directly proportional to the amount of dsDNA ONLY. As far as I can understand calculating the amount of dsDNA at the end of thermal run-start of kinetic run junction will be more direct here.

Sudha.

03/12/2013
Hi Sudha,

I and Raj have discussed about the extension kinetics results that were presented in the paper. Both of us have thought that using the data that were presented in the paper, it is possible to derive the extension rate parameter. I have raised few questions on estimating the Michaelis Meten kinetics parameters and gave my explanation on why should we plot the data differently. Raj will go through my explanations and get back to us on estimating the Michaellis Menten kinetics parameters. He will also comment on the possible experiments and simulations that can be performed using PCR machine to validate kinetic parameters. This experiments and simulations will also give some idea about the robustness of the estimated kinetic parameters.

In addition to this extension reaction part we need to do experiments on the Melting and Enzyme binding.

Our PCR simulation studies revealed that enzyme binding reaction is the rate limiting and hence, this will affect the overall kinetics of the PCR. The current enzyme binding kinetic parameters that we have used in our simulation is based on equilibrium thermodynamics data (Datta and Licati) of Taq enzyme binding reaction and our own assumptions. In order to validate our assumptions, we need to experimentally find the forward and reverse rate constant of enzyme binding reactions. I have given the above reference, Datta and Licati, which discuss the experimental procedure to determine the equilibrium constant of an enzyme binding reaction, based on Fluorescence anisotropy assay method. Can you go through the experimental section of the above paper and determine whether it is possible to conduct those experiments in PMC? Also, it would be nice if you can comment on conducting kinetics experiments using the above assay.

We have done the annealing study of the small primer molecules and I believe we are waiting for the absorbance spectroscopy method. Once we receive an update on the absorbance spectroscopy method, we can continue to work on this.

Finally, we will have to do some study to understand the kinetics of melting of long DNA molecules. Is it possible to determine the melting time for different length of DNA molecules (length more than 500 bp)? For example, we will consider a double stranded DNA and melt that for different times. Each time we will measure how much of double stranded DNA is converted in to single stranded DNA. Even if we can have only the time required for complete melting, that is sufficient. We will need this data at 90 deg or 95 deg C alone. At this temperature, we will assume that melting is an irreversible reaction with first order kinetics. So, if you can provide me the time required for the complete conversion of double strand molecules in to single strand, I can determine the rate constant. Note that here we will have to consider DNA with more than 500 base pairs. Please let me know whether it is possible to do all these experiments.

Thanks and Regards,
Karthik.

02/10/2013Hi Sudha,
I have read the document. I will discuss with Raj about your question on using the current data and will get back to you soon. I will also answer the question on the temperatures at which the experiments should be conducted.

Also, without converting RFU values in to concentration, we can fit the data if the following assumption is correct.

If there is a linear relationship between RFU values and the concentration of dsDNA in the form y = mx, then I can test the quality of the data based on the fitting I have explained.

Best,
Karthik.

02/08/13

Karthik,

Please find attached the word doc DNA annealing kinetics_experiments with my comments in it.

Sudha.DNA annealing kinetics_experiments KM update 020813 SM comments.docx

02/07/2013
Hi Sudha,
Please find the attached file for the next steps and questions on the experiments that were conducted to determine annealing kinetic parameters.
DNA annealing kinetics_experiments.docx
01/10/13
Karthik,
I understand your theoretical explanations.
Briefly, in protocol I:
90deg-70degC: decreasing temp., leads to annealing of complementary strands, leads to increase in fluorescent signal, which we see.
70degC incubn: over time, increasing annealing leads to increase in fluorescent signal, which also we see.
(The dip in signal happens after the first 2 cycles of the prolonged incubn (meaning start RFU of the 70deg incubn is the same as end RFU of the 90-70deg step) By cycle 10, the RFU starts increasing with increasing time and net gain in RFU over 2320secs is ~150 at 70degC and ~200 at 74degC. (By the way, the dip in the 80degC curve is even sharper almost 500RFU)
In protocol II:
90-65degC: decreasing temp leads to annealing of complementary strands, leads to increase in fluorescent signal, which I have recorded. These are single measurements and therefore cannot be plotted.
65deg-70degC: melt step, increasing temp, leads to decrease in fluorescent signal, which also we see
70degC incubn: over time, increasing annealing leads to increase in fluorescent signal, which also we see
(By the way, the expt was conducted till saturation, I do apologise the curve did not show the full extent. I have corrected the curve and am uploading a corrected version. Please take a look at the curves again).

(Note: Cycle 1 of the melt step shows a higher RFU than the 65deg RFU measured in step 2 (which is what I have taken as the start RFU for these curves), even tho’ we expect these to be the same).

My feeling about the steep dip we see in protocol I and the slight pick up in RFU in protocol II is that it’s a limitation of the CFX96 instrumentation software.
As I mentioned, I do not expect continuity in RFU values when we go from one loop in the PCR program to another or from a melt step to a loop. The software deals with each step separately creating separate curves for each step. The melt step RFUs are supposed to be raw but PCR step RFUs are not. Our 70deg prolonged incubn (which is neither a melt step nor a PCR step) RFUs are probably not raw (tho’ I would have to talk to a tech support person to be sure). By removing the option of baselining, I have tried to get raw numbers throughout, but as we can see there are some discrepancies.
Also the software is set for PCR reactions meaning, exponential increase in RFU numbers. It does not know how to deal with these small increases (I have had similar issues when I was doing the polymerase activity assays and measuring linear extensions on the PCR machine)
There is an essential difference between the two approaches (even tho the net gain in both approaches is not very significant given gains attained during PCR):
Protocol I: 90 to 70: annealing to equilibration (net RFU gain is ~150)
Protocol II: 65 to 70: melting to equilibration (net RFU gain is ~536)
It is likely given the software limitations one protocol works better than the other.
This is the reason, I tried the 2 protocols, to see which would work better.
And this is the reason I created the normalized curves. Since we are mainly interested in prolonged incubn at a specific temp, I made the start RFUs (of the prolonged incubn step) = 0 (slide 14), the Y-axis is then the delta RFU and directly gives the net gain in RFU.
When we get the protocol for the spectrophotometer, it should overcome all these issues because it will be specifically set up for a melt run (absorbance vs temp) followed by a kinetics run (absorbance vs time at a set temp) in one continuous process with continuous measurements and the absorbance and temp reads for every time point will be available. These will be actual reads without any processing. Additionally, since there wont be any dye in the system, we will not have to worry about the dye effect.
Please see the edited presentation for corrected curves and also (where necessary) more explanantions for your questions.

Oligopair A1a16-A2 annealing plots ver SM 011013.pptx

01/10/13

Hi Sudha,

Please find the attached presentation with questions and doubts. Please see the left corner in each slides (not in the slide show view) to view them.

Protocol 2 gives better understanding than protocol 1.

Please give me your comments. In the mean time I am doing some equilibrium thermodynamics calculations on this study. I will get back to you soon with this.

Thanks,

Karthik.

Some theoretical understanding:
We decrease the temperature of a reaction mixture where there are complementary DNA strands which can anneal from 900C to 70 or 750C (this temperature is decided based on the melting temperature of the given DNA strands). When the temperature is decreased the RFU value is increasing. We also know the time vs temperature profile and using this we should be able to find the RFU value at any temperature. The RFU values at these temperatures, however, may not correspond to the equilibrium concentration of the double stranded DNA at that temperature.

Once the temperature reaches a specific value , say 70 deg C, it is fixed and the RFU values is noted with respect to time. When we stop the increase in temperature at this temperature, the corresponding RFU value is not the equilibrium value at 70 deg C. If it is not equilibrium then the RFU value should increase when time increases.
For a given primer set, we can theoretically calculate the equilibrium concentration at any temperature and compare this with the experimental result to check whether the reaction reaches the equilibrium quickly or not.

Especially the above analysis is helpful for protocol 2 results. At any temperature the equilibrium concentration that we theoretically calculate should be lesser than the experimental results.

Oligopair A1a16-A2 annealing plots.pptx

Preliminary Results 01/04/13

Karthik,
Please see attached power point file for preliminary results for oligo melting experiments in the BioRad CFX96 Real Time PCR machine
Have tried to ensure that all relevant details are included, however if anything is unclear, please feel free to get back with questions.
Is this the kind of information needed?

Sudha.

Oligopair A1a16-A2 annealing plots.pptx

Experiment Planned for the week 12/17 - 12/21

To standardize a program on the BioRad CFX96 PCR machine for measuring oligo-annealing-time at a specific temperature.

End Goal: Set up an assay that will enable measurement of annealing time for two complementary strands of DNA (at a specific temperature) towards determining rate constants of the annealing reaction.

I will be using a pair of complementary oligos, 40bases long with a GC content of 50%, Tm = ~75oC. Following is the sequence of the oligos:
A1a16: 5'-GACTGCAAAGATGGAaACGACCTTCTATGACGATGCCCTC-3'
A2: 5'-GAGGGCATCGTCATAGAAGGTCGTTTCCATCTTTGCAGTC-3'

The reaction mixes contains both the oligos in equal molarity in a buffer solution, also containing SYBR Green I fluorescent dye. Samples containing no oligos are used as controls.

Steps:

Make the reaction mixes.
Heat the mixes to 90oC for 1:30mins.
Cool down to 75oC at the slowest possible rate while measuring fluorescent signal.
Keep samples at 75oC and measure fluorescent signal over time, say 5mins
Plot curves for fluorescent signal vs time at 75oC

The PCR program is as follows:
1 90.0 C for 1:30
2 90.0 C for 0:01
+ Plate Read
Increment temperature by -0.1 C per cycle
3 GOTO 2, 149 more times
4 75.0 C for 0:01
+ Plate Read
5 GOTO 4, 299 more times
END

Basically this is what it means:
Step 1: will denature the DNAs
Step 2: will read the fluorescent signal and then decrease temp by 0.1degC, then read signal and then decrease temp and so on 150 times (this should bring down the temp to 75degC).
Step 4 will do the same as step 2 except the temp will be maintained at 75degC. Measurement of signal will be every 1 sec for 5mins.

In this program, at any given cycle, we know the time from start of expt., also the the temp. of the sample. The fluorescent signal can be plotted against time/ temp/ cycle no.

This is the working plan. Please see next update for some preliminary results. Reaction conditions will need tweaking.

Sudha.

Hi Sudha/Raj,

Please find the attached file for the description of experimental work that need to be done.

I am also attaching the references here.

An approach to conduct kinetic measurements in real time PCR machine.docx

Stopped-flow kinetic method for the fluorimetric determination of DNA.pdf

CO-Operative Non-Enzymic base recognition.pdf

Relaxation Kinetics of Dimer Formation.pdf

Relaxation kinetics of the helix-coil transition of a self-complementary ribo-oligonucleotide A7U7.pdf

Salt dependence of DNA binding by Thermus aquaticus and Ecoli DNA polymerases.pdf

I would like to make a constant progress in this project and have a constant touch with the appropriate experimentalist.

Best,

Karthik.