RC (10/27):
A preliminary draft of the Research Strategy section of SBIR will be posted in dropbox folder shortly. It will continue to be edited but the tasks below should be addressed in the meantime:
1) Figures (numbers refer to those within draft)
n Fig 1: fix typos
n Fig 2: will need a caption indicating that generalization of PCR follows from OC-PCR’s ability (shown in Fig 1) to design cycling protocols for any objective and sequence. Some examples are shown; two-step PCR is another example
n Prepare a combined version of Figs 1,2 (Fig 1a,b) and assess space saved
n Fig 3: Title of Fig should indicate Sequence- and Temperature-Dependent Kinetic Model for DNA Amplification. Caption should explain that this model is an essential feature of the automated OC-PCR workflow (shown at right) that computes optimal cycling protocols for any PCR reaction
n Fig 4: Need to make sure pt a is legible; caption needs to indicate a,b) – caption for a can be taken from JCP paper 1. Need to make sure rate constant notation is explained (what are kf,kr - forward,reverse annealing rate constants, vs k1 – k1 is explained in text). Appears writing in a may not be legible – in that case may consider using picture in slide 26
Also, throughout all flowcharts, indicate whether the text is legible at the current figure sizes.
n Fig 5: May replace it with slide 41 (a,b) with a caption (based on JCP paper 2) indicating that the dynamical model for PCR can be used to predict the amplification efficiency of any PCR cycling strategy, including nonstandard strategies like the one shown here.
n Fig 5: have indicated to Alok to make an alternate version with more experimental data shown. May consider inclusion of rxn scheme in slide 32. If we don’t use it here, may use it in ppt presentations to other parties in the future
n Fig 6: May need to edit this to add the melting flowchart as posted. Details on this will be provided shortly. If you need to remake the Fig in order to do this prior to adding new features to the flowchart, please do so now.
n Figs 6,7: make their color scheme consistent with other flowcharts in the doc
n Fig 7: may indicate in caption that the algorithm identifies OC-PCR cycling protocols u*(t) such as those depicted in Fig 1.
n Fig 8: Would like this to show more clearly the cloud component of the workflow. The PCR machine client could be shown uploading sequence information to the cloud, followed by computation of OC-PCR protocols on the cloud, followed by machine downloading the solution and implementing it. The simplest modification would be to show that everything on the bottom row (blocks with text) are being done on the cloud.
Ppt will continue to be developed for presentations to other parties as well. Several of the tasks below are yet to be addressed and not all require further input.
2) References should be added to our 3 papers and when others seem to be referenced (e.g., in prior PCR modeling work), insert citations based on the bibliographies of our papers
3) For the work plan (section 3), we will be making a table of milestones that will make the timeline more explicit. Please consult with Sherry who may provide an example of how the table can be made.
This will later be connected to budget.
4) Miscellaneous: further info needed regarding the expiration dates of basic PCR and thermocycler patents - it is stated in slides when basic PCR patents expired in Europe; when in US? Also thermal cycler patent expiration date in US stated; Europe?
Please provide a rough estimate on when these tasks will be completed and updates on the points above as they become available.
The draft was posted to dropbox on Mon afternoon.
PL(12/29): Figures were prepared using powerpoint, Figures 1-3,5-8 are attached to the end of SBIR research strategy Phase I.pptx in the dropbox, Figure 4 and a combined Fig1&2 are in a separate ppt file. The jpeg version of figures are also posted. Color scheme in Figure 7&8 can't be changed as the original flowchart is not available.
I will continue to work on the figures and the rest of the tasks above.
RC: The figure on generalized PCR should ideally used that term; also, the OC-PCR protocol there is not really two step. I though in one of the academic ppts there was a depiction of two-step PCR. Perhaps it is too clumsy to add it, but please consider whether it is. If it is cluttered, perhaps we can just mention in the caption that two-step PCR is another example or otherwise indicate that COLD PCR is one representative example of modified cycling protocols.
Regarding 7,8, can't we remake the flow charts?
Please comment on readability of the font in these and other figs
Regarding melting fig, we may need to mention in caption that this procedure gives forward and reverse annealing rate constants.
PL(10/31): I have removed the two-step in figure 2 caption. (The word was chosen because it looks like some of the two-step PCR protocol described elsewhere.).
I will remake figure 7 & 8.
I have remade many of the figures. As most of the font size can be adjusted to be more readable, fonts in some of the flowchart may be too small to read due to the amount of text. In the cloud computing figure, the input interface may be hard to read as well.
I added the " forward and reverse annealing rate constants" in figure 4.
PL(10/24): An updated version of SBIR ppt is under Dropbox\PMC-AT PLIN\PCR_OPT\SBIR research strategy Phase I.pptx . Comments of a)-c), d),h), m) have been addressed. Need further instructions on some of the comments below.
RC (10/20): Comments on latest draft:
a) may add digital pcr (next-gen) to significance;
b) may move pcr variants slide to innovation;
c) may add another two-sided melting fig from jcp paper 1;
d) may show a transient kinetics fig from extension paper via alok and associated milestone 1b should come before full model slides; ask alok for it, it is in experimental extension paper draft (guan can direct him to the right document, and rc will then verify and send any recent draft updates)
e) need to add cloud computing and a couple of other application/product dev slides to conclusion (transition to phase 2);
f) good that extension estimation discussion is already included - testing required;
g) full pcr model should remain where it is with text later indicating that the model is now complete at end of phase 1;
h) may leave space for specific database that models are tested on;
i) insert captions for figs from papers where available;
j) cvi (oct algorithm and results) slides should be moved to algorithm section;
k) new slide should be allocated to new algoriithms to be implemented;
l) may indicate in slides that optimality conditions will be taken from working paper on pmc-at homepage;
m) milestone numbering should be fixed, last slide may be replaced w relevant phase II milestones;
n) preliminary results on oct should be put under milestones 2a,b) - not background;
o) citations should be fixed
p) may make a schematic for melting based on the procedure listed in BP, JCP papers and patent (do this after making progress on other tasks and providing the next ppt update - then indicate to RC when ready to start work on it, at which point further details will be provided)
q) may indicate w color coding how full model schematic will be augmented with parameterized melting model (together with p)
RC (10/13): Next steps for SBIR ppt:
a) the ppt needs to be revised based on the content we have allocated to Phase 1 and Phase 2. Most of the detailed slides and aims regarding applications, product and partnerships need to be
moved into a Phase 2 section.
The Phase 1 section will correspond to Research Strategy in SBIR proposal (6 pgs)
b) A few of the latter slides (most important high-level ones) can remain in Phase 1 at the end, in a section pertaining to next steps (one page of proposal, roughly).
c) The first section of the ppt on the patent landscape, etc should be titled Significance.
d) The core of the proposal pertaining to specific aims needs to have a corresponding section in the ppt. We need to order the slides in this section (which will be titled accordingly) in a way that corresponds to the detailed
aims and milestones. Note that most of the existing slides on the technology pertain to past work. Thus, just as in the specific aims we have summarized past work and then proceeded to mention how will complete certain tasks building upon that specific past work, we need to leave a blank slide, after every 2-3 technical background slides, that will later be filled in with bullet points pertaining to the details of the new proposed work and the associated milestones.
This needs to be divided into two sections, Innovation and Approaches. Innovation can contain the most important slides including the flowcharts for the models, and Approaches can contain remaining slides that go into more technical detail on the methods.
The bulk of the work required in ppt revisions will be dedicated to this task.
e) 3-pg draft of part of the SBIR proposal is available and will be provided once progress has been made on the above. The remainder of the proposal will then be written based on the template in the SBIR slides.
PL(08/22): The first draft for PCR presentation for SBIR is available under PMC-AT shared dropbox under PMC-AT PLIN/PCR_OPT/OC-PRC for SBIR_v1.pptx, or through the following link.
https://www.dropbox.com/s/7qak076vfuxfhnf/OC-PCR%20for%20SBIR_v1.pptx?dl=0
No all suggestions were made into the current version, but will be included.
PL(08/08): A second draft of PCR presentation is available on PMC-AT shared dropbox under PMC-AT PLIN/PCR_OPT/OC-PCR Presentation2.pptx , or through the following link.
https://www.dropbox.com/s/ty9kvl8uemy7m3k/OC-PCR%20Presentation2.pptx
Further modification is still underway.
RC: Please indicate under my latest points below which have been addressed and which have not/why. In other versions as noted in the original outline or below we will need to move some of the front end patent and COLD/digital PCR details to the subsequent sections since our technology is coming in too late otherwise. Regarding cloud computing, there should be a software diagram in my cmu ppt for decyded pcr algorithms that could be useful. After some more progress is made, I will advise on how a DNA melting flow diagram may be made based on patent claims.
PL(08/08): The final version of the Figures is now available on the PMC-AT shared dropbox, and can be accessed through the following link: https://www.dropbox.com/s/t5e8qi7tloklw04/CHARLOTTE-%23114279-v1-PCR_Enhancements_PCT_Figures_final.pptx
RC (8/8): It seems the flowchart for annealing is Fig 6, whereas I thought it was Fig 23 before. In any case, as noted based on discussion with lawyer we cannot change the order/numbering of the figures from what they were in the originally submitted ppt and summary section where the fig numbers are listed. The Fig numbers will need to be changed throughout the other portions of text instead (later). Hence I believe you need to fix the Figure numbers and order in ppt and make sure they are the same as original.
RC (8/20): A few more points about the ppt revisions:
a) there is much attention given to claims of previous patents but not to claims of ours. our patents should be portrayed as optimally controlled pcr patent portfolio (multiple patent applications forthcoming).
b) our patent claims can be highlighted for example as follows:
- minimal time optimally controlled pcr claim (this is a dae claim near the end of claim list) could come just after where the red bold font states that optimally controlled pcr could improve on rapid cycle pcr
- dna melting model claim and fixed time optimally controlled pcr claim (latter is also a dae claim near end of claim list) could come just after where the red bold font states that we could improve upon cold pcr protocols
The claims may need to be shortened/summarized in order to fit - we can discuss prior to doing this.
c) In the slide where we say none of other next gen PCR patents constitute dominant IP, we can provide more details. first, when we talk about real-time pcr, we should say that it is used to estimate the initial DNA concentration (i.e., for quantification), and relies on a standard curve to do so. Digital PCR provides a method for quantification without a standard curve. Since these methods cannot predict the dynamics/amplification efficiency of PCR for any given cycling protocol, they cannot be used to obtain new cycling protocols.
Target enrichment is one example problem where a new cycling protocol must be determined. COLD PCR offers a heuristic method for doing so. OC-PCR is general method for obtaining new cycling protocols.
d) the slide with two blue flow charts may need to come earlier
e) the slide on technology significance should be split up - the third point should occupy a slide of its own, whereas the first two might come early in the presentation when pcr is introduced
f) In slide 41 of v2, there a several ways to write the flowchart, we should use the following: under target DNA sequence, replace f(seq) with seq; under the arrow connecting the model box to the optimal control box, add J(u) and define it as amplification objective. Then replace f(seq) with J(u) in statements below.
g) Please add the patent application number (you can get it from Risa).
h) On slide 20, it says enter OC-PCR just before getting into many details of digital and COLD PCR. The placement of these bullet points is not appropriate given that digital and COLD details follow rather than precede this slide in the current version.
i) Around slide 83, prior to discussing diagnostics applications, there should be some bullet points regarding how we will now discuss how OC-PCR is a potentially dominant/general PCR technology by showing its various applications.
j) In the software section, we should indicate in a bullet point or two the following - now that we have demonstrated the generality OC-PCR, how can it be made available to that broad spectrum of users? In order to make it possible to use it in PCR machines engineered for any of the above applications, a cloud based platform is ideal.
k) BP paper is now in press; that can be mentioned.
RC (7-30): We are making some minor patent revisions required by the patent office. The first set of changes pertains to making minor corrections to the figures
that were submitted with the patent:
CHARLOTTE-#114279-v1-PCR_Enhancements_PCT_Figures.pptx
PMCFigs.pdf
There were some issues with these figures that were subsequently fixed (during the BP paper submission process and revisions to the manuscript "Dynamics and Control of DNA Amplification"). They need to now be made to the patent figures. These are:
a) fix legends on the minimal time figs (Figs 15-17). The times quoted were wrong. we can replace with the updated Figs 15-17 provided below.
b) Figs 7,8: check how difficult it is to change k1,k-1 to ke,k-e as the notations used in the patent text differ from those used in the figures (see the disclaimers in the figure legends). you have a copy of the patent text so you should verify what notation was used there. note that the latest BP notations (see BP_revision_final.pdf below) may not be identical to those in patent text, but we cannot change patent text notations; thus we can only adjust the patent figure notations to match the patent text notations. Please summarize the notations used throughout (comparing patent text to BP_revision_final.pdf) and indicate the changes you will be making to Figs 7,8 here:
RC: Have you compared the notations between patent_text and BP_revision_final? As noted above please summarize the notational differences.
c) compare the remaining BP figs to the corresponding figs in the patent. in the BP paper, there may have been some changes to the arrhenius plots for the rate constants that have propagated through some of the pcr simulation figures. identify
where these differences occur. we will then decide whether to replace the corresponding figs with the latest from BP.
I am attaching the latest BP submission here:
BP_revision_final.pdf
After your assessment of the differences between the figures in this pdf and the patent figures above, if we choose to replace any of these figures, I would need to give you the separate figure files. We are not required to do this; it is optional.
PL: The pdf file attached "BP_revision_final.pdf" is titled "Sequence-dependent biophysical modeling of DNA amplification", and it does not have the Figs 15-17 presented in the patent. I saw a pdf version titled "
Dynamics and Control of DNA sequence Amplification" on pmc-at website, do you have a updated version of that one,
I can fix Figs 7 easily, and can re-make Fig 8 without much difficulty. I will change k1,k-1 to ke,k-, which are the notations used in the patent text.
RC: Please comment on the differences in any/all notations between BP and patent. Also please analyze point c) today and post accordingly after a,b).
Replacements for Figs 15-17 (these are from the paper called "Dynamics and Control of DNA Amplification" posted on the PMC-AT website):
optimal_root_gg_new-eps-converted-to.pdf
opt_eta_gg-eps-converted-to.pdf
opt_temp_time_opt_ext-eps-converted-to.pdf
15-17 are here as noted. Do you need a different format? Here is eps
opt_eta_gg.eps
optimal_root_gg_new.eps
opt_temp_time_opt_ext.eps
I will let you know once you should start working on these changes, based on the deadline for submission. At that time I will post to the tasks page. In the meantime you may
have a brief look to understand the scope of the required changes and consider how the changes might be made (i.e. using which software tools).
RC (7-31): Ping, there is an upcoming deadline for this (middle of next week). Therefore, please work on the above tasks as a priority today and post by EOD your analysis of all of the above (a-c). (It is ok if the PCR ppt is delayed by a day as a result).
Did you read the comments above regarding 15-17 - they address your question.
PL: Here is something I observed. The Figure 15-17 were quoted as figure 16-18 in the patent text on page 32 right before EXAMPLE 3.. On the first sentence of the same paragraph,a Table 2 is quoted but not presented. Also, in the patent text, the three annealing time and temperatures are 30 deg C - 30 s; 40 deg C - 30 s; and 35 deg C - 120 s. which are not consistent with any of the figures supplied. What kind of adjustment should we make?
For request c), on Figure 11 in the patent, the k_-1 need to be changed to k_-e, and on Figure 12, k_1 need to changed to k_e as well. Other than these, I don't see much difference on the Arhenius plots between patent and BP.
RC: I will get back to you on the issues above after I check with the lawyer on what we can edit. The issue is that we may be only allowed to change the figures not the text (which is different from editing a paper submission obviously). In the meantime please provide answers to all the questions above including verifying whether there are -any- changes in any of the other BP figs vs the patent (not just the Arrhenius).
RC: I received the reply from the lawyer. It will be possible to fix typos in the text. Hence the figures should be corrected without constraints. The figures 15-17 above are the correct ones. Please propose your full list of changes to the figures and also reply to all points above (a-c).
PL: Here is a list of comments related to the content in Patent:
1) On page 11, Figure 12 provides estimates of ke temperature dependence.
RC: Please elaborate - what is the problem
PL: Not exactly a problem, just the wording may be a little confusing, because of the plot is Arrhenius plot of k_1 itself, and although it is calculated from k_-1 from last plot.
2) On Figure 4, The meaning of dash line data were not explained.
RC: Was it explained in the paper?
PL: The Figure is Fig 14 of the BP paper. The dash lines are calculated using the theoretically estimated hybridization rate parameters and solid line is from experimentally estimated hybridization rate parameters.
3) One of the Figure 5 should be Figure 23
4) Figure 5 was not found be to quoted before Figure 6
5) Figure 23 was quoted on page 21 before Figure 7
6) Page 25, the reaction (R4) has symbols missing
RC: Were they provided in the paper? If so, please indicate which symbols
PL: I believed this might be an issue of the word processing, they are not properly shown on the printed version I am having. many squares instead of symbols. Same issue below.
7) Page 32 second paragraph, “Table 2” was not presented but used. Figure 16-18 should be Figure 15-17.
8) Symbols missing in Eq. (29) on page 33.
RC: See 6 above
9) Figure 19, 20 correspond to Figure 18, 19
10) Figure 21 - 23 correspond to Figure 20 -22. However, the order of Figure 20 and 21 should be changed to be consistent with the description.
RC: These text issues will be passed to the lawyer along with a list I have been preparing. As a general comment, many of the the figure numbers were displaced by one in the text because a figure was added (note that the figure list at the beginning of the text matches the ppt, but the references to the figures in the text often do not, as you mentioned); so some of these figure numbering errors are part of the same issue.
The text does not need to be updated immediately. Regarding the figures, however - which are the priority - please note the tasks above and reply to all points requested by Fri am. I will review the ppt after that.
PL(08/01/): I have checked all the figures in the updated ppt file, and they are all consistent with BP papers.I did however, move the Figure 23 to the end, which should now move back to be #5.
RC: In Fig 7 there is ke,k-e but in Fig 8 there is k1,k-1. This should be checked/corrected along with the analysis above under b). b) could be done first so we can settle on the notation to be used in the patent. It may be advisable to make minimal changes to the text where possible so if there are no inconsistencies, we may choose to keep the patent text notations for rate constants as is.
Also, please check the resolution of all figures. They appear quite low. Is this because they are pdf not eps?
RC: Regarding consistency of the data presented in the figures (not notation), do you mean that the BP and patent figures show -exactly identical- simulation data? I am asking because I recall Karthik reran the simulations with some changes in the latest version of the BP paper. However, perhaps the changes were so minor they are undetectable.
PL: My mistake. On Fig 8. I re-make the flowchart in order to correct the issue, but forgot to make the change in the last step. The corrected version is not in place in the updated file.
The figures in the ppt files are indeed very low. If the figure in the pdf file is high enough, it is possible to convert them using the some graphics tools. I didn't make changes to those figures other than Figure 7-8, 15-17. If you can send me those figures in high resolution pdf
format like you provided above, I can create high resolution figures for you.
RC: Please clarify regarding the type of file you would need for high resolution figures, which figures you would need these for, and what type of conversion you refer to.
Please let me know if the figures in the attached file are identical to those in the BP_revision_final. If so, you can use these figures. For the annealing kinetics (JCP) figures, I have shared the dropbox. Please let me know if those figures match.
Please also reply to my latest point under b) above regarding comparison of notations.
Sequence-dependent biophysical modeling of DNA amplification_revision_071014_clean.docx
PL: As to data in Figure 15-17, they seem to be consistent with the latest "Dynamics and control of DNA sequence amplification" paper (see table 3 below). And other figures are consistent with those from JCP and BP_revision_final.pdf files.
PL(08/01): Here are the latest version of figures in ppt file. I kept the figures in high-resolution, and replaced those at lower resolutions. Let me know if you find any figure that need further adjustment.
PL(08/05): Attached is the updated ppt files with numbering based on the patent text. The last flowchart was not referenced in the text, but kept in case you decided to use it (should be before Fig 5). The notation in Fig 11 and 12 need to be changed according to your final decision on the notation to use. The ordering of Figure 20 and 21 was switched according to the patent text on page 36, in which temperature profile appears before DNA concentration.
CHARLOTTE-#114279-v1-PCR_Enhancements_PCT_Figures_PLv2.pptx
PL(08/05): Suggested changes to the patent text:
1) On page 8, line 3, subscript "r".
2) On page 11, renumber the Figures. Switch order of figure 5 and 6. Insert the original Figure 23 (now Figure 7) after Figure 6.
3) On page 11, Figure 12, replace k_1 with k_e, k_-1 with k_-e.
4) On page 13, change "Table I" to "Table 1".
5) On page 19, use Italic for k_1 specified; change Figure 6 to Figure 5.
6) On page 20, no Equation 11 prior to Equation 12, suggesting adding one before it. There are also equations not numbered on page 15 and 16, which can be numbered.
7) On page 21, add sentence " Figure 6 shows the flowchart for calculation of annealing rate constants." on line 6.
8) On page 21, change "Figure 23" to "Figure 7" on line 14. On section B, line 3, change "Figure 7" to "Figure 8".
9) On page 22, change "Figure 7" to "Figure 8" on line 10, change "Figure 8" to "Figure 9" on line 17. Also on page 22, check (R3) to make sure all displays are correct.
10) On page 24, line 1 and 7 of second paragraph, change "Figure 9" to "Figure 10".
11) On page 25, use subscript on cat and capitalized N on section 2) title. On line 2 of section 2), change "Fig. 10" to "Fig. 11". Double check the display of (R4).
12) On page 26, number the table as Table 3.
13) On page 28, change " Figure 11" to "Figure 12" on line 8. Change 6. to 6) for section title. Change "Figure 12" to "Figure 13" on line 5 of section 6). change k_1 to k_e on line 4 of section 6). subscript the cat and capitalized N on line 7 of section 6).
14) On page 29, change "95 C" to "90 C" on line 3, "Figure 13" to "Figure 14" on line 5, and line 5 from the bottom.
15) On page 30, change "Figure 14" to "Figure 15" in three places on bottom four lines.
16) On page 31, the temperatures and duration of annealing time need to be changed according to Figure 17, and a few occasions "C" need to be replaced with "degree C".
17) On page 32, Change "Table 2" to "Table 4" on the last paragraph of EXAMPLE 2. Insert Table 4 before EXAMPLE 3.
18) On page 33, check displays on Eq. 29, change k_1^e, k_-1^e to k_e, k_-e.
19) On page 36, change "Figure 22" to "Figure 21" as specified.
20) On page 44, subscript "-" on claim 23.
21) On page 47, change k_1^e, k_-1^e to k_e, k_-e in claim 32.
New figures are provided in the latest ppt file above. The BP version of Figure 14 and Figure 15 are attached at the end. If applied, changes need to be made accordingly on page 29.
RC (8/05): Did you add the sentences regarding the explanation of the dotted line to the bottom of the appropriate figure?
If we use the BP versions, as discussed, those will have to be edited - it appears you have used the versions from the old BP doc file?
PL(08/05): The explanation are now added to Figure 4.
As to the Figure 12 and 13 using BP notation, I haven't made them because it takes quite some time editing the figures, and it will cause a lot of changes in the patent text.
RC (8/05): Please change the notation in Figs 11 and 12 to ke,k-e, which is the notation we are using in the other figures. Also, please prepare an alternate version of the ppt with the flowchart as Fig 4 (since I believe that is where the closest associated discussion appears in the text?)
Also, please add to the end of this ppt two slides corresponding to the alternate versions of Figs 13, 14 from BP, in case we can use them and make associated changes in text.
PL(08/05): Can you share the folder for the BP paper that contain each Figures in high resolution? That will make things a lot more easier.
Also, the BP notations of k_1^e, k_-1^e, k_2^e, k_-2^e are probably better because k_-2 was used before in the annealing model, i.e. (R1, R2, Eq 5, Eq 6).
Regarding the figure where you said that there was no explanation of the dotted line, please include under the figure the closest text or caption from the associated paper draft that explains the dotted line.
RC: Are you sure there are no changes in Fig 14 with respect to BP_revision_final? Also, can you please fix the spelling of Threshold in Fig 14?
PL: The Figure 13-14 is consistent, the only thing may required a change is the labeling of the reaction time on Fig 14, which is better represented as in a range 30-90 s. However, I did not find the corresponding figures in the pdf files I am having.
RC: Regarding the reaction time labeling: isn't this figure an example of where the BP_revision_final figures differ from those in the patent? Is this why you say you don't have the corresponding figures (the 30-90s range is represented in BP_revision_final figs). So, do you confirm that these are the -only- simulation results that differ between the BP and the patent figures? (I would like to make sure that if we replace Fig 14 with a figures from BP, that these simulation results are not inconsistent with the other results presented in the patent. Otherwise, we may not replace Fig 14.)
PL(08/04): Figure 13 and 14 are not the same as those in the BP_revision_final paper, the simulation condition are different, both temperature and time. If you are about to replace the Figures, you will need to make changes in the text as well. Other Figures are the same as appeared in BP papers, except for the notation difference in Figure 7, 11 and 12.
I have already change the spelling of Threshold in Fig 15. See updated file above.
As to the notation between patent text and BP paper. They are mostly consistent. One place I found is (R3) on patent page 22, (R4) on page 25, which is actually in its correct notation form.
RC: I am pretty sure that in BP_revision_final.pdf, there are different rate constant notations compared to the patent. E.g., k_1^e, k-1^e, etc. Please comment on the extent of these differences with respect to the patent.
RC: This task remains
RC (8-4): Please reply regarding your comparison of the rate constant notations above Mon am.
PL(08/04): Yes. There are difference in notation about these rate constants. As I have pointed out above, in (R3) and (R4) of patent text page 22 & 25 (which actually should be R4 & R5), DNA binding is using k_e and k_-e, and dNTP binding is using k_2, k_-2, which is consistent Figure 7. That's why I said they are actually in its correct notation form. In BP paper, R3 and R4 using k_1^e and k_-1^e for DNA binding and k_2^e and k_-2^e for dNTP binding. But changing to the BP notations is probably better, but we need to change all these notations in the patent text as well Figure 7.
Also, in Figure 11 and 12, the notation k_-1 and k_1 need to be changed according to current patent notation (k_-e and k_e) or BP notation (k_-1^e, and k_1^e).
RC (8-4): Ok, please edit notations in Figs 11 and 12 to use k_-e and k_e (I believe you have the required figures in the doc file above). Regarding R3/R4 vs R4/R5 eqn numbering, please add this issue to your list of text issues above and if you find any other eq numbering issues, please add those as well.
There appear to be 2 figure 7's in PLv2 above.
Also, please check the notation on AxUx in Fig 1 to make sure they match the patent text.
PL(08/04): The numbering of Figures is not the final version because no reference of Figures in the patent text are not consistent with the Figures currently presented.
RC(8/04): Please edit the ppt Fig numbering so it is consistent with the proposed text numbering you have indicated above. It will be possible to fix the text numbering.
PL: Figure 1 is exact the same as in JCP paper, and there are two places in patent referencing Figure 1, pages 14 and 19. There is no problem with the referencing.
There is also a missing table 3 for page 32, which is attached here, but the text version need to obtain from original paper.
RC (7-20): Please provide an update on this as well as on the questions on the lead discovery report, along with a schedule of work on both.
RC (7-25): Please provide a rough draft of some of the slides today so I can have an idea of the progress. Thanks.
PL(07/25): The current PPT file contains the information collected so far.
Some significant additions include some important/milestone digital PCR patents.
OC-PCR Presentation1.pptx
One quick question: on slide 56 and 57, there is a drop in DNA concentration at cycle 27. Do you know is it an artifact or is it real?
RC: It is a numerical artifact due to stability of the algorithm. In the latest, BP paper, we have fixed this. I will provide the revised figures when you need them.
Regarding slide 45 - technology significance, are you going to include the paragraphs that I believe were provided?
RC (8-1): Some additional comments regarding the current ppt posted:
a) slides on previous (e.g. Higuchi) patents need to highlight the main claims
b) the slides taken from the pcr ip landscape ppt are probably too detailed to be readable (though may be useful for SBIR)
c) digital PCR significant IP slide(s) - similar, need to be streamlined for a readable presentation
d) the defs of cold and digital pcr (slides 29-30) may need to come before the digital and COLD PCR patent slides and possible
after "New variants of PCR cycling protocols"; order needs to be considered
e) new slide 35: just need to augment slide 37 so it refers to the appropriate amplification objectives corresponding to COLD, fast, etc
f) Technology Significance - as noted it has been provided; could be summarized in bullet pts
g) As in defense ppt, OC technology overview probably need some outline at the start to organize the parts of the model - annealing, extension, denaturation
Also, the flow diagrams from the patent/bp for each step may be included.
h) Are you sure there were no more slides on extension model in defense ppt?
i) For denaturation, we may include a summary of our corresponding patent claim where the steps are provided (can be done later)
j) For minimal time, there should be backup slides near end of defense ppt that show eqns for minimal time control. These can be provied
as a slide in appropriate place
k) Quotes regarding science have been provided
l) Title of paper 3 needs to be changed as indicated
m) Optimally Controlled PCR Patent slide can provide a summary of the main types of claims, perhaps by lifting some statements from the patent summary section (ask me for details)
n) Generality and IP position: fast, COLD could be provided as two examples, referring back to earlier slides. Further details on these follow
o) Sections 3 (typo): should start w a few bullet points on what applications will be discussed in this section. Here we are mostly introducing the goals of fast, COLD, digital (target enrichment, multiplex, e.g.) and then mentioning that the OC-PCR technology introduced above can be applied to the same problems. More technical details of the methods will come in section 4. Possibly, could move slide 90 before COLD and digital
p) May be too many details on COLD PCR in this section - may lose point. This is why we save more details on COLD, digital for Section 4, since we do not want to spend too much time explaining other technologies at risk of losing focus on our own
q) May remove comment about NEB
r) Digital PCR for multiplex may combine the "Digital PCR is emerging..." Will need a couple of bullet points on why it is good for multiplex and quantitative PCR (compartmentalization - no competition between primers, digital readout rather than quantification based on exponential growth curves)
s) Section 4: Here we go into more details on COLD and digital technology and very specific example problems. In SBIR or business plan, we would propose these as the concrete clinical applications that one could eventually apply OC-PCR to (the corresponding SBIR milestones could later be integrated in the appropriate place).
t) Section 5: For cloud computing, can insert slide on DecydEd/AFTP (previous PMC-AT cloud computing product)
u) Starting at slide125, there should be some introductory bullet points indicating that these are possible partnerships being surveyed. Associated SBIR milestones could be inserted.
v) There should have been a Fluidigm target enrichment slide where we can indicate OC-PCR can be used in such devices for deep sequencing enrichment.
w) Reagent slides - idea is that partnerships could be developed wherein particular reagents (e.g. polymerases) are marketed as being ideal for particular OC-PCR applications.
PL(07/22): Step one: Preparing ppt for the SBIR proposal and business plan.
Currently, collecting and converting text and pdf formatted information into powerpoint slides. Selecting and organizing information for the desired purpose.
RC (7-8): Ping, we have prepared a detailed set of slides (mostly from those previously prepared) and associated bullet points to be added, for the purposes integrated PCR ppt described below.
A folder is being prepared with all the materials. This folder will be presented to you. You will need to collate everything together, remove extraneous bullet points from the slides, add the new bullet points per the notes to the provided,
and make sure all slides are of the same, professional style and quality. Once you have returned from your trip, you should schedule work on this into your calendar. I am away all of next week, during which time you should start to work on this.
RC (7-11): Attached is an updated version of the optimally controlled PCR technology slides. You should look at the two ppts for optimal PCR, and replace the old slides in the folder provided to you with the corresponding new slides when making the new integrated ppt above.
Defense_04242014.pptx
RC (6-26): After cleaning of the optimal PCR technology ppt below, we will then proceed to prepare the draft overview ppt mentioned below that integrates slides
from several presentations.The first part of this ppt will be in the style of the PCR IP landscape ppt below, updating that presentation with some of the more recent
COLD and digital PCR IP and how companies are positioning themselves within that landscape. Then we will add optimal PCR technology, including accepted papers,
comments from others on the technology, filed patents (leading to multiple applications), discuss its applications to the problems mentioned below, the cloud computing
interface by which it can be used with any PCR machine, followed by some preliminary results or a proposal for how preliminary application-oriented results will be generated
with SBIR funding.
This ppt will be used as an outline for written documents/proposals.
RC (6-26): A short list (< 10) of "real-time quantitative PCR patents", especially those from Roche, should be included below. Risa, please do this.
Attached are four zipped files containing PDF's of patents.
Risa 6-11-14
The zip file below contains the following patents in PDF format:
1) Adaptive baseline algorithm for quantitative PCR US20030148332
2) Digital analyte analysis 8535889 Digital PCR
3) Methods for analyzing dna US20140113300 A1 Digital PCR
4) Methods for sequencing nucleic acid US20130210638 Digital PCR
5) Methods and compositions to enable multiplex cold-PCR US20140051087
Methods for sequencing nucleic acid US20130210638 Digital PCR.zip
The zip file below contains the following patents in PDF format:
1) Enrichment of a target sequence WO 2009017784 A2 Cold & Digital PCR
2) Full cold-PCR enrichment with reference blocking sequence US8623603
3) Microvescle-based assays US20140147839 A1 Cold & Digital PCR
4) Non-invasive mehod for diagnosing and monitoring glioma EP2586875A1 Digital & cold PCR
5) Step-up method for cold-PCR enrichment US20130309724
Non-invasive mehod for diagnosing and monitoring glioma EP2586875A1 Digital & cold PCR.zip
The zip file below contains the following patents in PDF format:
1) Kit and method for sequencing a target DNA in a mixed population WO_2012_118802_A1 Cold PCR
2) Methods for detecting multiple target nucleic acids US8697363 B2 Digital PCR
3) Methods of detecting diseases or conditions using circulating diseased cells WO 2013188828 A1 Cold PCR
4) Methods of detecting diseases or conditions WO 2013188846 A1 Cold PCR
5) Nucleic acid detection using probes WO2014077822A1 Digital PCR
6) Probe based nucleic acid detection US20130045881 - Digital PCR
7) Single cell nucleic acid analysis US8628923 PCR inadequate
Single cell nucleic acid analysis US8628923 PCR inadequate.zip
The zip file below contains the following patents in PDF format:
1) Analysis of nucleic acids by digital pcr US20090053719 Digital PCR
2) Apparatus and methods for parallel processing of micro-volume liquid reactions US20120077194 Digital PCR
3) Determination of fetal aneuploidies by massively parallel US8008018 Digital PCR
4) Digital pcr calibration for high throughput sequencing US20100069250 Digital PCR
5) Direct Molecular Diagnosis of Fetal Aneuploidy US8574842 Digital PCR
6) Methods for determining and inhibiting rheumatoid arthritis US8680066 Cold PCR
7) Methods and compositions for nucleic acid analysis US20120316074 Digital PCR
Determination of fetal aneuploidies by massively parallel US8008018 Digital PCR.zip
Attached is the list of patents for Cold PCR and Digital PCR.
Risa 6-10-14
List of Patents Cold Digital PCR 6-9-14.xlsx
RC (5-30):
Attached is a ppt surveying optimal PCR technology.
Defense_04222014.pptx
This document will have to be cleaned so it is not presented in the context of a thesis.
Also attached are some documents describing how PCR is used in next-generation sequencing (NGS), an important market.
PCR target enrichment for NGS.pdf
NGS_COLD-PCR.pdf
The list of ppts that will be used to create a draft overview of our product and its applications is:
a) The above ppt on our technology
b) Fast PCR overview
c) COLD PCR and digital PCR (below)
d) PMC-AT diagnostics talk (below)
e) Supplemental materials (such as those posted above).
We will also mention the applications of our optimal PCR technology to digital PCR machines, wherein we indicate possible advantages in terms of the modeling due to the fact that one does not have to model as many background molecules, given that each compartment contains fewer molecules, which are isolated from others.
RC (6-9): Here is a survey of the PCR IP landscape as of 2009. We will be updating this and including e.g. COLD and digital PCR patents.
Risa will be asked to do a patent search on these, then we will integrate summaries of these into a powerpoint that surveys the whole IP landscape. This IP review will be part of the presentation mentioned above.
pcr_ip_landscape.pdf
RC: The following link provides more info on real-time quantitative PCR vis-a-vis digital PCR:
http://www.ddw-online.com/enabling-technologies/p149031-dna-diagnostics-gets-digitised-fall-11.html
CJ 2/14/14
I will read the materials and then talk to Prof. Savran's students.
RC 2/14/2014:
Prof. Cagri Savran (Purdue) is interested in the possibility of collaborating with us in the area of mutation detection in circulating tumor cells (CTCs) and cell-free DNA in blood using COLD-PCR. He has a microdevice that is capable of isolating CTCs from blood and is asking whether we can do COLD-PCR to profile the mutations in these CTCs.
The principle is similar to that described below:
http://www.businesswire.com/news/home/20120904005304/en/Transgenomic-NYU-Collaborate-Lung-Cancer-Study-ICE#.UvpvRp6wJNN
These are rare circulating tumor cells collected from cancer patients' blood samples. They will be accompanied by some background (leukocytes). They will all be "fixed" in some formaldehyde solution.
I have asked Prof. Savran to provide a contact with whom CJ can speak to get further information required to determine whether we can run COLD PCR on samples the Savran group could send us. This would be a pilot study to assess feasibility. Ideally it could be carried out in conjunction with the COLD PCR experiments you were planning to run anyway following up on Sudha's work and the Poland algorithm calculations. It is an opportunity to get clinical samples we might otherwise not have access to. I will ask Savran to request his postdoc to email CJ to set up a time for a brief discussion where questions about the samples could be asked prior to committing to this study.
Q&A with Prof. Savran:
- Would there need to be innovation in the PCR or is there sufficient innovation in the CTC detection alone? For example do we need to show that in our PCR we can detect rarer mutations or mutations that are otherwise more difficult to detect in such samples?
Savran: An innovation in the PCR is desirable (I was assuming you guys have a better PCR) because there's a limit to how few CTCs you need to do PCR. The fewer cells you need (in a more complex background of other cells) the better.
RC note: Savran later indicated standard COLD PCR will be sufficient to start.
- Are you also interested in cell-free mutation profiling or just CTC mutation profiling?
Savran: We are interested in cell-free mutation as well but i though that could come later. E.g. we could do mutation analysis in CTCs and also in circulating free DNA, as well as in primary tumor.
- Are the mutation profiles going to be compared to anything? See e.g. the above. What do we anticipate doing with the mutation profile information? Are the samples from any particular stage of cancer? Are the patients being treated with anything? This is an important point since we can generate all this data but we should have an idea what we're planning to do with it.
Savran: We can get samples from any stage (I have collaborators in IU cancer center. we just added two GI oncologists to our team of collaborators). There are a number of applications. People are interested in CTC analysis before&after surgery. My big passion is 'assessment of response to therapy' and 'individualized treatment'. So with CTC analysis if we could look at very specific mutations, I'm thinking that would give the doctor personalized and specific info before prescribing drugs and/or changing their dose.
Savran: I think what's most important at this stage is to determine whether the number of CTCs we capture from a sample are enough for your system to detect mutations. That on its own would be a big deal because normally people need millions of cells for a PCR (which you will never get when hunting down CTCs: they're very rare). But I know it can be done with specialized PCR techniques (even with a single cell), but it's supposed to be expensive.
RC 1/29/2014
PMC-AT_diagnostics_talk_1-2b.ppt
An overview ppt of our strategy in PCR-based diagnostics is attached above. This should provide some background for the PCR tasks on Ping's tasks page, especially in terms of the types of markets our new technology is addressing and how reduction of cycle time or increase in the selectivity of amplification of a mutated sequence can impact those markets. However, this is not a comprehensive list, since software that is capable of kinetic modeling and control of PCR (and oligonucleotide hybridization) can broadly impact the DNA amplification industry. Also, some of the results are outdated, with the BP paper draft having more recent results. The current focus is on PCR modeling, primer design and PCR optimal control software, not the solvent/media engineering techniques mentioned early in the ppt.
This is just one document in the "PMC-AT Research/Diagnostics" dropbox folder. That folder has more background material.
CJ 12/27/2013
Attached below are (1) a report on Tm simulation using Poland algorithm; (2) the original data used to generate the curves, in spread sheets; and (3) the reference for Poland algorithm.
The Poland algorithm is a free online tool: http://www.biophys.uni-duesseldorf.de/html/local/POLAND/poland.html
Poland Algorithm for cancer mutations.ppt
Poland Algorithm.xls
nar00038-0052.pdf
CJ 12/2/2013
I have three things in hand right now:
(1) Overall editing of the paper, 'aim to finalize the paper including all formatting so it can be submitted without those sections if needed'.
(2) Prepare a plan on the beta-lactamase project and start to work on it.
(3) prepare a list of important cancer mutations. To do this I will need to comb through the COLD-PCR literature.
Any of these three tasks would need full attention. My plan is to finish (1) by the end of tomorrow, then work on (2). The BL21 cells should arrive within a couple of days, and I'm starting the bench work soon. If I could find some spare time during the bench work, I will try to work on (3). But it is hard for me to give a promise on when I can finish it. Please let me know if you prefer some other order of priority.
CJ 11/26/2013
A summary of comments on today's group meeting:
(1) We are not going to repeat other people's work. We will develop new diagnosis technologies.
(2) Sudha's work on melting curve may be patented.
(3) We are going to do some fundamental research on domain melting. Hopefully that will help us to develop PCR method to detect trinucleotide repeat expansion. - Study domain melting of these repeats via MELTSIM, for various repeat lengths, determine whether the melting curves can be reproduced experimentally, then obtain melting curves in presence of solvents experimentally. Determine at what repeat length denaturation is responsible for the decrease in PCR efficiency through melting curve analysis. If denaturation does not limit amplification efficiency in presence of solvent for a particular repeat length, possibly publish results with emphasis on possible use of these solvent protocols in diagnostics without use of triplet repeat primed PCR. If problem lies in secondary structure that causes polymerase to dissociate, possibly characterize these structures either through computational prediction of secondary structure or experimental characterization of the fragmented products for several repeat lengths, in absence and presence of the solvent.
<this project needs further planning of publication strategy before additional work>
(4) We are also going to study the mechanism of heteroduplex melting at Tc, trying to predict and optimize enrichment fold of COLD-PCR. - use MELTSIM to predict domain melting Tm's and then compare against HRM.
We need to pay special attention to the effects of the mutation on the melting of the domain that contains the mutation. MELTSIM should be run on all mutations listed under (7) with this in mind. This will result in a shortlist of mutations that can benefit from greater enrichment than achieved through the standard COLD PCR protocol. PL can help with this if it is useful.
(5) We will use optimal control theory to optimize COLD-PCR. - PL; kinetic control of enrichment, decrease of cycle time
An issue with the conventional COLD-PCR protocol that could in principle be addressed with our control approach: 8 minute cross hybridization at ~70 C of followed by melting at 1 degree below Tm. These are the 2 new steps in COLD-PCR. This is an empirical protocol that does not have a clear quantitative foundation, unlike conventional single-sequence PCR protocols. For example, from a thermodynamics standpoint, equilibration at 1 degree below Tm will eventually lead to a fraction of the homoduplexes melting as well - given sufficiently long time at this temperature, the effects of the 70 C cross hybridization will be reversed and the distribution of species will be determined by the equilibrium melting thermodynamics at 1 degree below Tm. Thus the optimal cycling protocol for the (2) enrichment steps is not necessarily that used in COLD-PCR. The objective is to maximize the ratio of mutated single strands / wild type single strands at the start of the primer annealing step. Eventually, RC/PL can frame this problem quantitatively and solve it.
(6) We can also study how organic solvent affect mutant enrichment in COLD-PCR. -- narrowing of melting curves for homo and heteroduplexes to be assessed via HRM
(7) As a priority on the COLD PCR project, prepare a list of important cancer mutations along with their Tm's and Tc's and check the overlap of their melting curves via MELTSIM. Determine which ones will display lower enrichment factors (approximately calculate the enrichment factors) and shortlist them for further study, with goal of increasing enrichment factors. This is a mutation-specific enrichment protocol (need to know mutation in advance; this is an area where COLD PCR can be improved). Mutations and their clinical significance can be listed here.
CJ 11/20/2013
Here I'm attaching an updated version of the slides for the group meeting. Slides 63-79 on repeat expansion detection were newly added.
COLD-PCR and digital PCR.pdf
RC: Please summarize some of the discussion points from today's meeting regarding sensitivity, so we have a record.
It appears the statement that mutations need not be known in advance for COLD PCR applies to enrichment not detection.
RC: Also, I would like to see RC's original diagnostics presentation posted here.
CJ 11/20/2013
Please see my comments below. Here I'm attaching an updated version of the slides on COLD-PCR and digital PCR for the group meeting. Slides 9 and 10 are newly added.
COLD-PCR and digital PCR.pdf
CJ 11/18/2013
Please see my comments below.
Please find an updated presentation attached below. The ppt file was more than 20M and therefore can not be uploaded on wiki. If needed I can transfer it via Dropbox.
Slides 16, 19, 32, 50, 55-60 in the file below were newly added.
COLD-PCR and digital PCR.pdf
RC: A few follow ups: a) please add one slide on the MELTSIM issues; b) please comment on what is responsible for the greater sensitivity of digital PCR, given that it is often billed as an absolute quantitation technique; c) is there any info available on how the homo/heteroduplex Tm difference affects the efficiency of COLD PCR? d) why is an 8 min 70 C step needed - there may be room for improvement here; d) there was a typo on the last slide, it should say "minimize wild type DNA concentration".
CJ: a) I added it. It is posted above.
b) Here by saying sensitivity, I refer to the enrichment of mutant vs wt. COLD-PCR can pull out a mutant out of 1000 fold excessive mutant. Digital PCR can pull out a mutant out of 1000,000 fold excessive mutant. This is the intrinsic difference between the two methods. In other words, the COLD-PCR can enrich mutant to some extend, but there is a limit. Please also be adviced that the '1 out of 1000' sensitivity for COLD-PCR is actually for fast COLD-PCR only. The fast COLD-PCR relies on a significant difference in the Tm of the mutant vs wt and therefore is only applicable to certain mutations (preferably G>A). For other kind of mutations you have to use full COLD-PCR, which can pull out a mutant out of 200 fold excessive mutant.
c) I have not seen any of such discussion. I can keep on searching but it will take time.
d) Please see slide 5. The 70C is to form the homo/heteroduplex. They did not explain why it takes so long, but I assume the 8min is optimized based on experiment.
d) I fixed it.
Let's finalize the group meeting slides over the next day or so and then move back to the extension paper until the group meeting.
CJ 111513
Attached is a report by Sudha on 04-16-2012 on COLD-PCR. This is by far the most comprehensive report I've found on this project.
COLD-PCR Preliminary Report 041612.docx
Here are some preliminary slides I prepare for the literature group meeting, focusing on COLD-PCR and digital PCR for cancer mutation detection. I will prepare a separate file on the Fragile X project.
COLD-PCR and digital PCR.ppt
RC: Here are some items to highlight/expand on in the presentation:
Overall: emphasize the advantages/disadvantages of COLD vs digital PCR - esp the main problems with each.
CJ: First of all, these two technologies are not exclusive to each other. As I have mentioned in the slides, the COLD-PCR is for enrichment; the digital PCR is for detection. Actually I include one example in the slides that uses COLD-PCR in combination with digital PCR. Based on literature, digital PCR seems to be much more sensitive than COLD-PCR: the former can detect 1 mutant out of 10^6 wt; the latter, when paired with sequencing, RFLP, Taqman, HPLC as detection methods, can detect 1 mutant out of 10^3 wt. I will add a comparison table of these two tools.
COLD PCR: -what are the typical reaction times and number of cycles for the whole PCR? does one need to run more cycles than in traditional PCR?
CJ: I will check the literature and include in the slides. My intuition is that the time for COLD-PCR is comparable to regular PCR.
-what issues did Sudha face? these may be representative
CJ: First of all, I have not figured out a general picture on how much work she has done on this project. The progress report I posted last Friday was the only one I can find. Everything else are just scratches of figures and numbers - without a context they make little sense to me.
Talking about her progress report, basically she tried to reproduce a COLD-PCR experiment that was published before, but failed. She ran the COLD PCR followed by HRMA and a gel to check the product. No HRMA data was provided in that report, and the gel shows a smear (no image in the report). This may mean that the PCR did not work, neither the wt nor the mutant was amplified. Based on this one single report it is also hard for me to figure out what's wrong with her experiment. We need follow up information. Meanwhile I will also take a closer look at her report and try to see what I can find.
-what we can do - as may be mentioned in our earlier diagnostics slides, the COLD PCR protocol may not be optimal at distinguishing a specified mutant pair, and also may not do so in the shortest time. E.g., how does one choose the temperature to which the system is cooled to cross hybridize? What is the effect of overlapping melting curves between hetero and homoduplexes on the discrimination? To what extent does COLD PCR rely on a big low Tc? Some of this might be improved via kinetic control. We can improve on this with the predictive computational PCR models we are developing. You can hence include some selected slides from the earlier PCR diagnostics talk that describes how our computational PCR optimization methods may improve cycling strategies on a mutant pair-specific basis compared to COLD PCR (some of these slides might also be used to introduce our overall goals in PCR-based diagnostics).
CJ: I'm curious about how close is our kinetic control model to be applied to optimize a real PCR reaction? Has anyone ever used the model to successfully optimized a PCR reaction before? I'm not an expert on kinetic model or optimal control. But my intuition is that one needs to start from a proof-of-principle system, then push to real world application. The COLD-PCR, in my opinion, is a quite complicated system that is much harder to model and optimize compared to the regular PCR. So before we apply our model to COLD-PCR, I suppose we will start from regular PCR, trying to optimize the program based on the kinetic model. If that works, we will then move to COLD-PCR.
RC: Yes that's right. No one has done optimal control of PCR based on kinetic models before. Mutant enrichment is more complicated for sure, but is a "killer app" of optimal control of PCR in the area of diagnostics.
-is it true that mutant amplification rate in regular PCR is decreased in later cycles, but not substantially in earlier cycles?
CJ: I've never seen discussion about this. I will try to find some clue in literature.
-please comment on the use of MELTSIM to predict Tc and whether there are issues with that; i.e., does one need to determine Tc experimentally
CJ: For all the paper's I've read, the Tc were always determined by experiment. The MeltSim software can be used to generate a melting profile which is used to confirm that the amplicon has only one melting domain. It can also be used to predict Tm, but most people still determine Tm experimentally because it is very fast and straightforward, at no extra cost. After determining Tm, Tc is usually estimated as 1C lower than Tm. But the exact value is still to be confirmed by experiments. Basically the software is usually used as a guideline to get started. The exact Tc needs to be fine-tuned based on experiment.
Digital PCR: - seems to primarily be an absolute counting (quantitation) method (operating through spatial separation; the separation must done before signal amplification; due to spatial separation, it does not need to alter mutant/wild type ratio in order to detect mutant) that does not rely on exponential growth analysis. What aspects of the detection system result in it having greater sensitivity than COLD PCR, if indeed that is the case? I.e., assuming one is interested in detection, not absolute quantitation, what are its advantages?
CJ: As I have mentioned above, the digital PCR is much more sensitive compared to COLD-PCR in terms of mutant detection.
- if one could do absolute quantitation based on exponential growth (e.g. in COLD PCR) would there be any need for digital PCR?
CJ: It depends on how sensitive you need it to be.
- what are some typical uncertainties in quantitation based on exponential growth and why are these not acceptable in cancer diagnostics?
CJ: Regular qPCR quite accurate. And as far as I know, it is widely accepted in cancer diagnostics. The digital PCR does not offer a big improvement in terms of accuracy, it is for sensitivity.
However, if you uses COLD-PCR to enrich the mutant, you will not be able to quantify the percentage of mutant. You can only find out the mutant/wt ratio after enrichment, not in the original sample.
- what are typical reaction times and number of cycles required to get a detectable signal in each droplet?
CJ: The program is generally the same as regular qPCR.
- seems to have problems with multiplexing - e.g., how does one distinguish a mutant from wild type?
CJ: I did not see any problem with multiplexing in literature. You can use Taqman probe to distinguish mutant and wt as in qPCR. (Please see slide 12 for how Taqman works)
- why does the mutation need to be known in advance?
CJ: you must know it in order to design the Taqman probe.
COLD-PCR can be used to amplify unknown mutants. However, you will need to sequence the product in order the detect unknown mutants. TaqMan and RFLP can not detect unknown mutant even when used together with COLD-PCR.
- does anyone discuss the situation where there are both mutant and wild type in same droplet?
CJ: Two different Taqman probes, one for mutant and one for wt, are added together to all droplets. If one droplet has both mutant and wt, it will show signals in both mutant and wt channels (the two probes have different fluorophroes). Because the sample is well-partitioned, even both mutant and wt are in the same droplet, the wt/mutant ration in one droplet should be much much lower than that in the original sample.
- is there anything discussed regarding the number of polymerases in each droplet?
CJ: I will check about it.
- cycling is completely standard. We are most interested in digital PCR as a benchmark against which to compare novel cycling protocols like COLD PCR, which do not separate species, and do quantitation directly on a mixture. Our primary goal is to see if digital PCR is dominating the industry to an extent that renders it difficult for other technologies to compete - and if so, in which regard (sensitive detection, quantitation or both; certainly, it appears to offer superior quantitation)
**We may also consider presenting some of the extension paper work at the next group meeting as part of the talk. This will depend on where we are with that paper and how much time it would take to present the above. The extension work could be presented as one example of the predictive PCR modeling that can be used to develop optimal cycling protocols on a template specific basis.
CJ: I heard that the group meeting is scheduled on Nov. 26th. And Sherry told me the group meeting is of higher priority compared to the paper. I will concentrate on making slides at the moment, and see how much can be done on the paper before the group meeting.