CJ 11/04/2013
I can also take a look at the COLD PCR project. Would you please let me know where can I find Sudha's previous reports etc.?
RC: They should be on her computer and important materials can be reposted to wiki.
Here I am providing a presentation given to New England Biolabs (who have also licensed our patents) when they visited PMC-AT for a potential diagnostics collaboration. This is an overview of all our work related to diagnostics (repeat expansions, cancer, etc) and how it connects to the chemical and optimal PCR technology. For the group meeting, it should be augmented with a section on digital PCR and the goal would be to compare COLD PCR to digital PCR for the same mutation to understand the state of the art in early stage cancer mutation detection and the scope for improvement either in time, cost, sensitivity, etc. After that meeting you can be in charge of arranging a digital PCR demo from Biorad (filling out the appropriate demo forms, etc) that would ideally be run with the same primers and sequences used in the COLD PCR experiments so we can directly compare them.
rc_diagnostics_talk.ppt

CJ 11/01/2013
Below are several key references, including research paper and patents, on Fragile X and C9orf72 mutation detection. I can do a literature review group meeting covering all these and some more papers on this topic. Currently I'm curious about our specific aims, in order to do literature research with higher efficiency. As Raj has mentioned, commercial diagnostic services are already available for both Fragile X and C9orf72 mutation. According to the patent, it seems that these diagnostic services are mostly based on chemical PCR and CE technologies. I suppose we are not simply repeating these published methods (although we may re-validate some of them in the beginning). So do we have a general idea on what will be the key innovation?

RC: Yes, I had a literature review group meeting in mind as well. If you do this, you should include a survey of Sudha's work on COLD PCR and the possibility of redoing those studies with digital PCR, since cancer diagnostics (ultimately using our optimal PCR technology under development) is an equally if not more important goal for us in this area. I have a presentation we previously delivered to New England Biolabs on diagnostic applications of our PCR technologies that could be use in this regard.

This is a protocol paper on Diagnosis of Fragile X using PCR and CE with lots of experimental details. This protocol should also be useful for C9orf72 mutation detection.
Capillary Electrophoresis for the Detection of Fragile X.pdf


This is the patent by Quest Diagnotics on detecting CGG repeat in FMR1 and FMR2 genes, using chemical PCR and CE. This is probably the patent behind XSense. However, they did not cite any patent by Princeton.
WO2011097503A2.pdf

RC: We are currently being paid royalties for our patents on chemical PCR by Quest, and prior to that, by Celera, who indicated they were using it in their Fragile X test. Quest bought Celera, and shortly after that we began receiving royalties from Quest. It is probably from Fragile X XSense sales, though we cannot be 100% certain.


This is another patent on detecting CGG repeat in FMR1 gene. It uses a different method based on qPCR. No CE needed.
WO2010127020A1.pdf


This is a representative paper on C9orf72 mutation detection, using repeat-primed PCR and CE technologies.
A Hexanucleotide Repeat Expansion in C9ORF72.pdf


These are two patents on C9orf72 mutation detection, both based on repeat-primed PCR. One uses CE and DNA sequencing for detection, the other used CE, agarose gel, and DNA sequencing for detection.
WO2013036833A1.pdf
WO2013041577A1.pdf


RC 10/21: Please look into the C9orf72 gene, which undergoes a hexanucleotide (rather than trinucleotide) GC rich repeat in more than one neurodegenerative disease. It is one of the more promising markers in the area and could be studied side by side w Fragile X. Please do a literature search to see what is published regarding the latest tests marketed by Quest Diagnostics. Also please do a patent search to see for the Quest Fragile X and C9orf72 tests, what patented technology (e.g., capillary electrophoresis) is needed to run these tests. The XSense test from Quest uses at least our patents (which are owned by Princeton now). The goal would be to see if enough is published to allow us to run these tests. We do not have a capillary electrophoresis instrument, but perhaps that can be outsourced. We should check whether the C9orf72 test requires capillary electrophoresis as well. Previously (below), we have not been able to properly PCR (and analyze the PCR products for) the Fragile X gene. We may try C9orf72, but only after determining whether the literature reports successful PCR with enough detail to reproduce.
We might consider a group meeting on this literature at some point.
This diagnostic project can also involve increasing one's familiarity with the COLD-PCR studies Sudha did.


SM 8/26/13 C:

The Rd 1 PCRs were performed in Feb'13. Reaction samples were stored at -20degC, Rd2 has been started in Aug'13.

For the FT primer pair:
Only Taq leads (as per document in SM 8/26/13 A:
Strategy Forward Starting February 2013
) gave products in Rd 1 PCR. DV and Herculase Rd 1 PCR leads did not give a PCR product (see summary Table in SM 8/26/13 B).

Rd 2 PCRs for Taq Rd 1-pdt trials 1-3 did not give a positive reaction. Rd 2 PCR with DV for Taq Rd 1 PCR-pdt has given an amplicon of the target size. This result needs to be confirmed.

See attached files for summary of FT Rd 2 PCRs to date:
081613 Rd 2 NA20230-FT-Taq-N+B trial 1.pdf
081913 Rd 2 NA20230-FT-Taq-N+B trial 2.pdf
082013 Rd 2 NA20230-FT-Taq-N+B trial 3.pdf
082213 Rd 2 NA20230-FT-Taq-N+B trial 4.pdf

The following is planned:
1. Confirmation of Rd 2 PCR with DV for Taq Rd 1 PCR-pdt
2. FP2-RP2 Rd 2 PCRs using Taq as per denat-anng-ext conditions detailed below (L4-L7).
3. FP2-RP2 Rd 2 PCRs using DVas per denat-anng-ext conditions detailed below (L4a-L7a).





SM 8/26/13 B:

The following table summarizes the results at the time of writing (Aug' 13):

Lead	Primer pair	Conditions	Denat-ann-ext temp	Comments
L1	FT	Taq + 0.5M NMP +2.2M Bet	92-62.6/57.8-72	To be followed up in Rd 2
L2	FT	DV + 1M TMSO		No Signal
L3	FT	Herculase +0.5M NMP + 2.2M Bet		No Signal
L4	FP2-RP2	Taq +0.5M NMP	76-68.8-74	To be followed up in Rd 2
L5	FP2-RP2	Taq + 1M TMSO	76-70-72	To be followed up in Rd 2
L6	FP2-RP2	Taq + 1M Formyl Morpholine	83-68.8-75	To be followed up in Rd 2
L7	FP2-RP2	Taq + 0.5M TMSO	79-70-75	To be followed up in Rd 2
L4a	FP2-RP2	DV +0.5M NMP	87.7-57.8-71.6	To be followed up in Rd 2
L5a	FP2-RP2	DV + 1M TMSO		No Signal
L6a	FP2-RP2	DV + 1M Formyl Morpholine	75-57.8-73.9	To be followed up in Rd 2
L7a	FP2-RP2	DV + 0.5M TMSO	79.1-70-59	To be followed up in Rd 2
L4b	FP2-RP2	Herc +0.5M NMP		To be attempted
L5b	FP2-RP2	Herc + 1M TMSO		To be attempted
L6b	FP2-RP2	Herc + 1M Formyl Morpholine		To be attempted
L7b	FP2-RP2	Herc + 0.5M TMSO		To be attempted

At this point, SFS primers have not been tried at all.

For the FP2-RP2 primer pair only Taq leads (L4-L7) were suggested. These leads (L4-L7) have been tried, also based on these leads, DV reactions (L4a-L7a) were tried. Similar herculase reactions (L4b-L7b) need to be tried.

Please note that with FP2-RP2 primer pair, several bands are usually obtained, that are also very close in size, thus it has not been possible to identify the correct target band. What we have are “possible” target bands based on approximation of size with the molecular ladders we’re using.

The following is planned:

1. 1. Check reaction components and order if necessary
2. 2. Start Rd 2 PCRs first for Taq-FT primer pair (L1), followed by Taq-FP2-RP2 (L4-L7), DV-FP2-RP2 (L4a-L7a).
3. 3. Simultaneously perform Rd 1 PCR for Herc-FP2-RP2 (L4b-L7b).

See attached files for summary of Rd 1 results.
The Rd 1 PCRs were performed in Feb'13. Reaction samples were stored at -20degC, Rd 2 has been started in Aug'13.

The following are results for FT primer pair-Rd 1 PCR with Taq
NA20230-FT-Taq-N+B denat grad 75-95.pdf
NA20230-FT-Taq-N+B anng grad 50-70 rpt.pdf
NA20230-FT-Taq-N+B ext grad 55-75 anng 62.6.pdf
NA20230-FT-Taq-N+B ext grad 55-75 anng 57.8.pdf

The following are results for FT primer pair-Rd 1 using Deep Vent:
NA20230-FT-DV-TMSO denat grad 75-95.pdf

The following are results for FT primer pair-Rd 1 using Herculase:
NA20230-FT-Herc-N+B-Bet denat 75-95.pdf


The following are results for FP2-RP2 primer pair- Rd 1 using Taq:
NA20230-FP2-RP2-Taq-NMP-For.Mor-TMSO denat grad 75-95.pdf
NA20230-FP2-RP2-Taq-NMP-For.Mor-TMSO anng grad 50-70.pdf
NA20230-FP2-RP2-Taq-NMP-For.Mor-TMSO ext grad 55-75.pdf

The following are results for FP2-RP2 primer pair- Rd 1 using Deep Vent:
NA20230-FP2-RP2-DV-NMP-For.Mor-TMSO denat grad 75-95.pdf
NA20230-FP2-RP2-DV-NMP-For.Mor-TMSO anng grad 50-70.pdf
NA20230-FP2-RP2-DV-NMP-For.Mor-TMSO ext grad 55-75.pdf

As mentioned above, at this point, SFS primers have not been tried at all.



SM 8/26/13 A

Following is the methodology currently being followed for developing a PCR protocol for amplification of the triplet repeat region of the FMR1 gene:
Strategy Forward Starting February 2013

[Note: Amplification of CGG Repeat Region of the FMR1 gene has been one of the toughest PCR amplification problem ever encountered. Accordingly, it is time for taing a hypothesis-based systematic approach to solve the problem. Shot-gun approaches for quick results would be detrimental to solving the problem. As you conduct the recommended experiments you will face several failures. The negative results are not bad. They often are as valuable as the positive results. The negative and positive results, only when they are viewed together, can help us reinforce and or modify our hypothesis]

Upon review of several published papers and our initial experimental results, we are now ready to put forward a tentative hypothesis and a FMR1 amplification strategy for the CGG repeat region in pre- and full mutation cases. The basic tenets of this hypothesis are:

1. Eliminate Hot-Start. The conventional PCR strategy, where every attempt is made to fully denture the Target DNA, would be detrimental in this case. The primary reason is that CGG repeats with more than 30 repeats without any AGG interruption are prone to form secondary structures such as hairpins and the Tms of most of these structures are higher than the Tms of the primers. The polymerases will tentatively pause when they reach the sites of these secondary structures. When the secondary structures are on the growing template, there will be deletions with resultant formation of lower length products. But when the secondary structures are on the substrate strand there will be slippage with resultant formation of longer length products. Hence Hot-Starts, particularly in formulations that contain additives/solvents (to reduce Tm), should be eliminated from the amplification strategy.

2. Establish Additive Systems and Cycling Conditions that are Specific to the Primers Used. In discouraging complete denaturation of the dsDNA, we suggest a novel PCR strategy, where we seek to melt (denature) only the primer-binding sites and conduct a strand-replacement extension process. Our goal here should not be to melt too long a dsDNA stretch (that can promote secondary structure formation) but a long enough stretch for the primers to comfortably enter the melted region and anneal to their respective complementary sites. Such a strategy will require primer-specific PCR formulations and cycling conditions that are also specific to those formulations (including the polymerase). This means that for every primer pair and polymerase there should exist a particular combination solvents/additives and cycling parameters that are specific to that system alone. A one-size-fits-all strategy will fail. This means that simply because an additive system (such as 0.5M NMP and 2.2M Betaine) worked on one set of primer/enzyme combination under one particular set of cycling conditions, that it will be good for another primer/enzyme system, where either the primer set or the enzyme or both are different. Our strategy should be to define those compositions and cycling parameters for the three sets of primers we have been working with --- namely the FP2/RP2 set (each 21 base long, with 81% GC and Tm ~75 oC); the FT primers (each 30 bases long; 57% and 67% GC respectively in the forward and reverse primers; and Tms 73 and 78 respectively); and the SFS primers (forward 28-bases long, 57% GC, Tm 73o; reverse 26 bases long, 65% GC, Tm 75o). We need to determine specific PCR formulations/conditions for each of these three primer pairs, since they represent binding sites that are at different distances from the CGG repeat region. We may need all the three primer pairs in later parts of our strategy for amplification of the FMR1 expansion sites. However our primary focus initially will be the FT primers.

3. Use Long Extension Time. Since strand replacement extension rates can be slower than those that represent uninterrupted extensions on a ssDNA strand, we would need much longer extension times during the PCR cycle. An extension time of two and a half minutes seems reasonable.

4. First Part of our strategy will consist of selecting a few leads for the three primer pairs -- L1-L3, L4-L7, and L8-L9, respectively for the FT pairs, P2 pairs and the SFS Pairs respectively. These Leads are based on our prior experiments and are shown in Tables 10 to12 below. The steps of optimization of each of the leads in tables 10, 11, and 12 will consist of the following.
a) Run a denaturation gradient between at 75-95 oC [78-98 oC in case of Deep Vent Polymerase] (30 sec) keeping the annealing and extension temperatures fixed at 54 oC (1.5 min) 72 oC (2.5 min) respectively. From this choose the optimum denaturation temperature that gives highest specificity for the target. Irrespective of whether the T band is weak or strong, the most important criterion for selecting the best T band is that it is the most dominant band. If more than one denaturation temperature give similar results, chose the lower one within the range. This step has already been done for the Table 10, L-1 lead (choice denaturation temp here was established to be 92 oC for Taq). Note that the weak band in L-1 could be due to instability of the Taq at the higher temperatures (92-95 oC) in the presence of the additives. Hence the recommendation is to try both the Herculase Fusion II and the Deep Vent polymerases as well [Table 10, L2 and L-3].
b) Now keeping the denaturation temperature fixed at the one chosen above (for 30 sec) and the extension temperature fixed at 72 oC (2.5min), run an annealing gradient between 50 oC and 70 oC. Chose the best annealing temperature using the same criteria as in (a).
c) Next run an extension gradient between 55 oC and 75 oC (2.5 min) keeping the best denaration temp (for 30sec) and the best annealing temperature (for 1.5 min) as determined above. Choose the best extension temperature using the same criteria as in (a).

5. The Second Part of our strategy will consist of running a second round of PCR for the best compositions and cycling conditions (for the three primer pairs) as determined in step 4 (c) above and using a 1:10 and 1:50 dilutions of the products. The idea here is to make the Target amplification as robust as possible and also to improve its specificity. If this step improves specificity but not enough, you may run a third round. First try to use the same enzyme in the second and third rounds, but you may also use a more processive enzyme like the Herculase Fusion II or the Deep Vent Polymerase if needed in these rounds.

6. Depending on the results obtained from the above experiments, we will decide if another step, like amplification of the products from the above strategies using a different pair of inner primer, will be necessary or not.

Selecting the Leads:

Table 10: Viable Leads for FT Primers + NA 2030 gDNA [Target band is 383bp]

Lead #	Polymerase	Solvent	Deanaturation	Anneal/extnd	Result	Comments
L-11	Taq	0.5M NMP + 2.2M Betaine	92-95o (better at 92o)	54 (1.5m) + 72o (2.5m)	Medium to Weak2 T	Quite specific.
L-23	Herculase Fusion II	0.5M NMP + 2.2M Betaine	Not determined. To be test at 75-95 oC gradient.	Same
L-33	Deep Vent	1.0 M TMSO4	Not determined. To be test at 78-98 oC gradient.	Same

1This is Sudha’s Expt #S46 (run 1-29-13) with a denaturation gradient (75-95o) using fixed temperature annealing (54 oC) and fixed temperature extension (72 oC). It is a follow-up of #S4 (Page 10-IV of Sudha’s report) except for elimination of HotStart.

2Weakness of the band may be due to poor stability of the Taq at 92-95 oC. Hence recommendation for

L-2 and d L-3.

3These experiments have not been run yet.
4The recommendation of 1.0M TMSO is based on L-5 experience (see Table 11).

Table 11: Viable Leads for FP2/RP2 Primers + NA20230 gDNA [T band is 221bp]1

Lead#	Polymerase	Solvent	Deanat	Anneal/extnd	Result	Comment
L-42	Taq	1M NMP	80 oC	60 oC	T (sp): medium	AE65 oC: faint (non-sp) AE45-50 oC: No T
L-5	Taq	1M TMSO	85 oC	65 oC	T (sp): medium	Good tolerance to AE 60 oC and 70 oC also works
L-6	Taq	1M Formyl Morpholine	85 oC	70 oC	T(sp): medium	AE65 oC also works but not as good
L-7	Taq	0.5M TMSO	90 oC	70 oC	T (sp): medium

1The above data were derived from Sudha’s experiments in 2011. These experiments were conducted using two-step amplification cycles. Ram ran the same primer systems but not under exactly the same conditions.

2Ram ran this composition (#R8) using hot-start (98 oC, 2min) and a two-step cycling process with denat at 85 oC and ann/extn at 50-70 oC. Got nothing.
Table 12: Viable Leads for SFS Primers + NA20230 gDNA [T band is 401bp]

[We do not yet have a viable lead for the SFS Primers run without hot start. What we have are two reactions one with Taq/0.5 M NMP and the other with Deep Vent/0.5M TMSO both run with a 98 oC hot-start that gives hope that more robust lead can be developed without hot start.].

Lead#	Polymerase	Solvent	HotStart	Denat	Anneal/ Extnd	Result	Comment
L-81	Deep Vent	0.5 TMSO	98oC, 2min	95 oC	50-74oC	Md T AE 66-74 oC	Higher + Lower bp products. No <66 oC
L-92	Taq	0.5 NMP	98 oC, 2m	85 oC	50-70 oC	Md T 50-70 oC	Twice successful-once failed2.

1This is Ram’s Expt# R12 run on Sept 10. Was not repeated.
2This experiment was conducted by Ram three times -- R16 on Sept 11, R21 on Sept 13, and R25 on Sept 18. He succeeded the first two times, but failed in the third. Sudha also conducted a similar experiment (S34, page 1-II of her report) but failed to get any product. Sudha’s experiment was with 0.5 NMP and 2.2 Betaine & without Hot-Start (S36 Page 28 of her report) – not the same as Ram’s. Gradient experiments will establish the correct cycling conditions for 0.5M NMP (without Betaine).