We are working on pioneering a novel detection method that leverages enzymes crafted from single-stranded DNA, specifically DNAzymes. Our goal is to create a platform that can detect DNA and RNA sequences with an exceptional level of specificity, down to the resolution of single nucleotide changes. This innovative platform is primarily designed for the accurate identification of unique sequences, especially in the context of pathogen detection. Additionally, we are exploring its potential for advancing the field by developing organic logic circuits, showcasing the versatility and broad applicability of this groundbreaking technology.

We have included the methods used by us for conducting these experiments below.

PCR reaction -

The template DNAZymes FBS1, FBS2, and FBS3, initially present in 100mM concentration, were amplified through the symmetric Polymerase Chain Reaction (PCR) process. This amplification was done using specific forward and reverse primers, allowing for the targeted replication of the desired DNA sequence. For three separate DNAzymes a total of 3 primers were used. The CommonSR primer, which serves as the shared reverse primer for all three templates, and two particular forward primers—FBS1S/FBS2S for FBS1 and FBS2, and FBS3S for FBS3

The PCR reaction mixture components included:

Template	1μl
Forward primer	2.5 μl
Reverse primer	2.5 μl
dNTPs	1 μl
Taq polymerase	0.5 μl
Taq DNA buffer	6 μl
Nuclease free water	17 μl
Total	30 μl

Initial Denaturation	Denaturation	Annealing	IInitial extension	Final extension	Hold
95° C	95° C	55° C	72° C	72° C	4° C
1 min	10 sec	30 sec	20 sec	2 min	∞

Gel Electrophoresis for confirmation of PCR Product:

1. Measured 1 g of agarose.
2. Agarose powder was mixed with 100 mL 1xTAE in a microwavable flask
3. Microwaved for 1-3 min until the agarose is completely dissolved.
4. Agarose solution was allowed to cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins.
5. Ethidium bromide (EtBr) was added to a final concentration of approximately 0.2-0.5 μg/mL .
6. The agarose was poured into a gel tray with the well comb in place.
7. The gel was kept at room temperature for 20-30 mins, until it had completely solidified.

Loading Samples and Running an Agarose Gel:

1. Once solidified, the agarose gel was placed into the gel box
2. Fill the gel box with 1xTAE (or TBE) until the gel is covered.
3. 100bp molecular weight ladder was loaded.
4. Samples were loaded into the additional wells of the gel
5. The gel was run at 80-150 V until the dye line was approximately 75-80% of the way down the gel.
6. Turn OFF power, disconnect the electrodes from the power source, and then the gel was carefully removed from the gel box.

Visualization of gel

1. BioRad gel viewer used.
2. Gel placed into the viewing platform.
3. Gel drawer closed and the gel manipulation window opened.
4. Alignment of gel done using incandescent light setting into appropriate visualization box.
5. Protocol for EtBr selected.
6. Run protocol.
7. Picture analyzed using inbuilt gel analyzer.

The PCR product was visualized on a 2% agarose gel containing ethidium bromide for size confirmation, utilizing a 100bp DNA ladder as a molecular weight marker.

PCR product purification :

1. 500μl of binding buffer was added to a MCT containing the PCR product
2. Then it was added to a purple column (Monarch® DNA Cleanup Columns)
3. Centrifuged it for 1 min at 14000 rpm.
4. Passed the flowthrough again to the purple column.
5. Centrifuge it for 1 min at 14000 rpm.
6. Discard the flowthrough
7. 500μl of wash solution was added to the purple column.
8. Centrifuged for 1 min at 14000 rpm.
9. Discard the flowthrough
10. Again 500μl of wash buffer was added to the purple column.
11. Centrifuged for 1 min at 14000 rpm.
12. Flowthrough was discarded.
13. Dry spined the purple column for 3 mins at 14000 rpm
14. Kept the purple column in new MCT
15. 30μl of preheated elution buffer was added in it
16. Kept for 5-10 mins
17. Centrifuged for 3 min at 14000 rpm.

3. Asymmetric PCR

After the initial PCR amplification of the stock DNA, an asymmetric PCR was conducted using the purified PCR product. The purpose of this asymmetric PCR was to selectively amplify a specific region of interest, focusing on one DNA strand. In this process, a single primer was used, designed to anneal precisely to the target region of interest. This primer served as the initiation point for DNA synthesis, facilitated by a DNA polymerase enzyme. This led to the preferential amplification of a single DNA strand.

The PCR reaction mixture components included:

Loading Samples and Running an Agarose Gel:

1. Once solidified, the agarose gel was placed into the gel box
2. Fill the gel box with 1xTAE (or TBE) until the gel is covered.
3. 100bp molecular weight ladder was loaded.
4. Samples were loaded into the additional wells of the gel
5. The gel was run at 80-150 V until the dye line was approximately 75-80% of the way down the gel.
6. Turn OFF power, disconnect the electrodes from the power source, and then the gel was carefully removed from the gel box.

Visualization of gel:

1. BioRad gel viewer used.
2. Gel placed into the viewing platform.
3. Gel drawer closed and the gel manipulation window opened.
4. Alignment of gel done using incandescent light setting into appropriate visualization box.
5. Protocol for EtBr selected.
6. Run protocol.
7. Picture analyzed using inbuilt gel analyzer.

The PCR product was visualized on a 2% agarose gel containing ethidium bromide for size confirmation, utilizing a 100bp DNA ladder as a molecular weight marker.

GEL extraction:

1. 500μl of binding buffer was added to a MCT containing the PCR product and kept at 65 °C till the gel properly got mixed with the binding buffer
2. Then it was added to a purple column (Monarch® DNA Cleanup Columns)
3. Centrifuged it for 1.5 min at 14000 rpm.
4. Passed the flowthrough again to the purple column.
5. Centrifuge it for 1.5 min at 14000 rpm.
6. Discard the flowthrough
7. 500μl of wash solution was added to the purple column.
8. Centrifuged for 1.5 min at 14000 rpm.
9. Discard the flowthrough
10. Again 500μl of wash buffer was added to the purple column.
11. Centrifuged for 1.5 min at 14000 rpm.
12. Flowthrough was discarded.
13. Dry spined the purple column for 3 mins at 14000 rpm
14. Kept the purple column in new MCT
15. 30μl of preheated elution buffer was added in it
16. Kept for 5-10 mins
17. Centrifuged for 3 min at 14000 rpm.

Qubit fluorometer is measurement to get ssDNA conc

1. A mastermix of the QubitTM working solution was prepared.
2. For calibration, the Qubit ssDNA kit needs two standards.
3. Give the tube lids labels.
4. To each tube, aliquot the QubitTM working solution:
5. QubitTM working solution and Sample tubes filled to 180–199ul
6. Each tube must have a final volume of 200 l after the sample and standard have been introduced..
7. Vortex each tube for 3 to 5 seconds
8. Two minutes of room temperature incubation .
9. "Read standards and samples" should be performed on all tubes.
10. On the QubitTM 3 Fluorometer's Home screen, tap DNA, then choose 1X ssDNA HS as the test type.
11. Close the cover after placing a sample tube within the sample chamber and push the Read tube.
12. Remove the sample tube after the reading is finished (around three seconds).
13. The original sample concentration is shown at the top (in large type), and the dilution concentration is shown at the bottom.
14. Thoroughly document all outcomes and save the Qubit's run file to a memory stick

CD spectroscopy

1. The purified DNA samples was diluted to make a solution of 200ul with concentration of 0.2mg/ml
2. The quartz cuvette was filled with the buffer solution used for dilution. This served as a reference or blank.
3. The reference cuvette was inserted into the CD spectrophotometer and performed a baseline correction by measuring the CD signal at around 200-300 nm.
4. Now the quartz cuvette was filled with diluted DNA sample, inserted in the CD spectrophotometer, the spectrum was recorded and the data was analyzed

Concentrating DNAzyme

1. Samples pipetted (200μl) in 1.5ml Eppendorf MCT
2. Samples balanced and kept uncovered.
3. Concentrator plus from Eppendorf used at 60 °C using Function V-AQ
4. Run at setting for 30 minutes uncovered.
5. 15μl of concentrated DNAzyme obtained
6. Same protocol, run on dsDNA, primers with same initial concentration.
7. Take a part of concentrated samples and dilute to 25mM in 200μl
8. Send all diluted samples for CD spectroscopy to check for quality after concentration.

Methylene blue staining:

1. Measured 1 g of agarose.
2. Agarose powder was mixed with 100 mL 1xTAE in a microwavable flask
3. Microwaved for 1-3 min until the agarose is completely dissolved.
4. Agarose solution was allowed to cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins.
5. Ethidium bromide (EtBr) was added to a final concentration of approximately 0.2-0.5 μg/mL .
6. The agarose was poured into a gel tray with the well comb in place.
7. The gel was kept at room temperature for 20-30 mins, until it had completely solidified.



Sample preparation

1. The 5μl PCR products were mixed with 5μl of methylene blue (used to stain ssDNA)
2. The solution was kept for 30 minutes.

Loading Samples and Running an Agarose Gel:

1. Once solidified, the agarose gel was placed into the gel box
2. Fill the gel box with 1xTAE (or TBE) until the gel is covered.
3. 100bp molecular weight ladder was loaded.
4. Methylene blue stained samples were loaded into the additional wells of the gel.
5. The gel was run at 80-150 V until the dye line was approximately 75-80% of the way down the gel.
6. Turn OFF power, disconnect the electrodes from the power source, and then the gel was carefully removed from the gel box.

Making DNAzyme action solution

1. Take Nuclease free water (we made a stock solution of 100ml)
2. Add Magnesium chloride powder (20.33g) to the 100ml solution
3. Add Sodium Chloride (5.488g) in the same solution
4. Dissolve and use when setting up DNAzyme-loop reactions

Fluorescence Reading

1. Load the sample in the platform
2. Keep the platform to incubate at room temperature for 30mins
3. Switch on the Fluorescence reader, and go to protocols
4. If TamR protocol is present then use the same
5. Otherwise, make a new protocol for tamR with excitation at 550nm and emission at 578nm
6. Load the platform
7. Run the protocol and take a reading
8. For multiple readings of the plate, re-run the protocol again and again at different time intervals to get as many readings as needed