PHY3904–PhysicsandAppliedPhysicsLaboratoryII

1

DNAHybridization/MeltingLabMeasurementofDNAhybridization/meltingcurves

‐Experiments designed in the Godin Laboratory by Jason Riordon and Michel Godin

1 Contents2 OBJECTIVES ........................................................................................................................................... 2

3 INTRODUCTION ..................................................................................................................................... 2

4 THE EXPERIMENT .................................................................................................................................. 3

5 THE DETAILS .......................................................................................................................................... 6

5.1 Fluorescence ................................................................................................................................. 6

5.1.1 SYBR Green I .......................................................................................................................... 6

5.1.2 Optical setup ......................................................................................................................... 6

5.1.3 Samples and Sample holder (Cell) ........................................................................................ 7

5.1.4 Theory and predictions ......................................................................................................... 7

5.2 Data acquisition ............................................................................................................................ 8

5.2.1 myDAQ card .......................................................................................................................... 8

5.2.2 LabView program .................................................................................................................. 8

5.3 Temperature sensing and control ................................................................................................. 9

5.3.1 Temperature monitoring circuit ........................................................................................... 9

5.3.2 Temperature control circuit .............................................................................................. 109

5.3.3 Temperature calibration ..................................................................................................... 10

5.4 Photodiode Signal Amplification ............................................................................................. 1110

5.4.1 Amplification ................................................................................................................... 1110

5.4.2 Photobleaching ............................................................................................................... 1211

5.4.3 Lock‐in Amplification........................................................................................................... 12

6 DATA ANALYSIS ............................................................................................................................... 1312

7 REPORT ................................................................................................................................................ 13

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PHY3904–PhysicsandAppliedPhysicsLaboratoryII

4

b) Material

To build your experiment, first take a look at what you have to work with. You should, amongst

other little things, have the following:

Custom temperature controlled sample holder/cell

Optical breadboard

SR540 chopper and controller

SR510 lock‐in amplifier

BK Precision 1698 switching mode DC regulated power supply

Thorlabs DET110 photodiode

Linear polarizers

Thorlabs LIU003 blue LED array

Chroma D470/40x excitation filter

Chroma E515lpv2 emission filter

NI MYDAQ data acquisition card

TCS‐620 Thermistor

Box of electronic components

Solderless breadboard

Thermometer

4 microcentrifuge tubes/samples

c) Procedures

1. You should start by assembling your setup. The sample holder should be secured at one

end of the optical breadboard and the light source should be aimed towards the cell and

secured at the opposite end of the breadboard. The heating elements should be inserted in

the cell. The photodetector should be mounted as to collect the excited light from the cell,

at a 90o angle with respect to the incoming excitation light. Two polarizing filters are to be

mounted (later, not for calibration) between the light source and the sample. Rotating the

filters with respect to one another will allow you to attenuate the excitation intensity. The

optical chopper will modulate the excitation light at a precise frequency, when used with

the lock‐in amplifier. The chopper and the lock‐in amplifier do not have to be used during

calibration, but it might be useful when making the actual measurements on the DNA

samples. Optical filters are used at the output of the light source and at the input of the

photodetector to isolate the relevant wavelengths.

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PHY3904–PhysicsandAppliedPhysicsLaboratoryII

6

the actual sample temperature is likely to be different than the measured cell temperature,

you will need to calibrate this.

5. DNA melting. With your DNA sample in place, you will expose the sample with the

excitation light and start ramping up the temperature. The provided Labview program can

record the output fluorescence intensity during the experiment. Details on this

measurement are included below. Please take note of the fact that the excitation intensity

needs to be reduced by about 75% in order to avoid damaging the dye through

photobleaching. This intensity reduction needs to be done prior to exposing your DNA

sample to light.

5 THEDETAILS

5.1 Fluorescence

5.1.1 SYBRGreenI SYBR Green I is a fluorescent dye that preferentially binds to dsDNA, and emits very little in free

solution. SYBR Green I emits at 497 nm, and absorbs at 520 nm. SYBR Green I exhibits a strong

dependence on binding, but also on temperature. During heating, you should observe a drop in

background fluorescence; during subsequent cooling, you will notice a rise in the fluorescent signal. In

your measurements, you will notice a linear dependence between fluorescence and temperature; this

should be subtracted out of your signal or can be ignored once you differentiate your signal to obtain

the melting temperature.

5.1.2 Opticalsetup To filter out unwanted light, you’ve been given two filters. Fasten the excitation and emission

filters on the LED array and DET110 photodiode, respectively (Fig. 3). The right angle geometry between

emission and detection helps reduce unwanted background. Be careful to handle the optics with care.

Position the photodiode directly on the temperature control cell to maximize collected light. Place the

LED array about 20 cm away from the cell. Stick the first polarizing sheet adjacent to the emission filter,

and place the second on a rotating stage in the beam path. By rotating the 2nd polarizer, you will be able

to easily adjust illumination intensity.

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PHY3904–PhysicsandAppliedPhysicsLaboratoryII

11

(connected to the handheld multimeter) through the hole, all the way to the bottom of the water‐filled

vial. The idea is to create a temperature table, to accurately convert your thermistor measurements into

actual sample temperature (it is not practical to use this manual thermocouple during an actual

experiment). So, ramp the temperature to 90°C, hold for 30s, and let it cool. All the while, note in an

excel spreadsheet thermistor and thermocouple values. Here, you assume the manual thermocouple

reading is close to the actual sample temperature. You will later use this calibration to convert the

thermistor reading to infer the actual sample temperature.

5.4 PhotodiodeSignalAmplification

5.4.1 Amplification Build the amplification circuit shown in Fig. 8. The Photodiode circuit is internal to the DET110,

and fully shielded. Be careful to attach the BNC to your circuit at the correct polarity. The T‐network

amplifier circuit is chosen to allow for a high gain current‐to‐voltage conversion, while keeping resistor

values similar.

We recommend you use resistor values of R1=100 kΩ, R2=1 MΩ, and R3=1 MΩ. While you can choose

different resistor values, you should aim for an output signal (for the fluorescein sample and NO

polarizing filters in place) to be in the 5‐8V range. Be careful not to exceed 10V, as the myDAQ is

susceptible to damage. Use the oscilloscope to monitor the signal before sending your signal to the

myDAQ card. Capacitors C2, and C3 are there to clean up the power supply signal, sending high‐

frequency noise to ground. Capacitor C1, on the other hand, will act as a low pass filter if you choose the

value properly. You should discuss your choices of resistor and capacitor values. Note: Be careful

plugging in the electrolytic capacitors: they’re polarized and can explode if improperly used. The trade‐

off here is the more you filter you signal, the slower your RC time constant and response time.

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PHY3904–PhysicsandAppliedPhysicsLaboratoryII

13

You’re ready to go. Run the program, and you should see a real‐time intensity signal, as well as the

thermistor measurement you set up earlier.

Of course, you could run your experiments without using the lock‐in amplifier. It would be interesting if

you discussed the differences between running the experiment with and without the lock‐in.

6 DATAANALYSIS

Acquire the melting/hybridization curves for samples labelled (i) and (ii) over 5

heating/cooling cycles. One contains the perfect match sequence; the other the single base

mismatch (table I). Can you determine which is which based on the melting temperatures?

Make sure to use you calibrations to compensate for temperature lag and offset, and

obtain a fluorescence vs temperature graph during heating and during cooling.

Use smoothing tools to clean up your melting curve.

Fit a Gaussian peak to the derivative, and extract the central melting temperature.

Compare your results to those predicted using theoretical models. If you fit your own data

to these predictions, you can also infer the thermodynamic properties of the DNA

annealing reaction, including ΔH°, and ΔS°.

7 REPORT

In your report, compare how melting temperature varied with both sequences. Can you tell which

sample was the perfect sequence, which was the mismatch? Are the measured melting temperatures as

expected? If not, what factors could have influenced this?

Your report should also discuss issues you have encountered during the experiment. You should

certainly discuss the steps you took to address these issues, or at least propose potential solutions as

future experiments.

Finally, please include a section suggesting potential improvements to this laboratory so that we

can improve the experiments in the future. You input is essential and much appreciated!