DNA Melting Part 1: Measuring Temperature and Fluorescence

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20.309: Biological Instrumentation and Measurement

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DNA melting apparatus block diagram showing the five functional groups of the instrument.


In theory, there is no difference between theory and practice. But, in practice, there is.

Jan L. A. van de Snepscheut/Yogi Berra/and or Chuck Reid

You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:

  • excitation
  • heating
  • fluorescent detection
  • temperature measurement
  • data acquisition and control

A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, LCGreen Plus and a salt. LCGreen Plus fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.

The dye is excited by blue light, which will be delivered by a low power LED. The dye emits green light. A photodiode will be placed at 90° to the LED source to detect the green light emitted by LCGreen Plus. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.

The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (f represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.

The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks.

Mechanical and thermal components

Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from ThorLabs. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.

  • 1 MB1224 optical breadboard
  • 2 CL5 clamps (or similar)
  • 2 1/4-20 5/8" screws
  • 4 1/4-20 1/4" screws

The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.

Parts

Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:

Center Cabinets The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.
Wet Bench Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.
East Cabinets The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.
  • These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it
  • 3 wire nuts
  • 1 Diablotek 250 W power supply (at your station or on a shelf above your station)
  • 1 red/black TEC extension wire assembly (in a box on the 'Center Cabinets')

Assembly instructions

TEC showing hot side up when current flows into the red lead; i.e., when a positive voltage is applied between the red and black leads.
  1. The bracket, TECs, fan and heatsink should already be assembled for you.
    • The TECS are sandwiched between the heating block (on top) and the heat sink (underneath).
  2. Verify that the colors of the leads match. If not, flip one TEC with reference to the picture at right.
  3. Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.
  4. Connect the TEC to the black/white (and black/yellow) extension wire assembly using wire nuts to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC, respectively.
  5. Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.
    • Use only the black 2x2 connector, not the white one.
  6. Test the TECs by touching the base of your heating block and turning the Diablotek on.
    • The base should start to feel warm within 10-15 seconds.
    • If the Diablotek immediately shuts off (the fan will stop spinning) then there is probably a short. Check all connections.
  7. Once the TEC assembly wiring is checked, try the heating test again.
  8. If it fails again, find an Instructor.

Optical subsystem

A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the Instructor instrument(s), and the instructions below, as a guide. The set of instructions can help if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode.

  • 2 PH2 post holders
  • 2 BA1 post holder bases
  • 2 1/4-20 1/4" screws
  • 2 1/4-20 5/8" screws
  • 2 1/4" washers
  • 4 CP02 cage plates
  • 2 8-32 1/2" setscrews
  • 2 TR2 posts
  • 6 ER3 cage rails
  • 12 4-40 1/4" setscrews (or similar length)
  • 4 SML05 lens tubes
  • 4 SM1LTRR retaining rings
  • 1 blue LED PCB
  • 1 D470/40 excitation filter
  • 1 E515LPv2 emission filter
  • 1 SM1A6 adapter between SM1 and SM05 threads
  • 1 SM05PD1A mounted photodiode

Assembly instructions

Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.

  1. Attach each PH2 to a BA1 using 1/4" screws.
  2. Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.
  3. Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.
  4. Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.
    • Leave the blank hole on the bottom of the assembly.
  5. Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.
  6. Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.
  7. Arrange on the MB1224 and adjust CP02 heights as desired.
  8. Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.
  9. Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.
    • The arrow label on the thickness of the filter points toward the light source.
  10. Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.
  11. Thread the photodiode into the SM1A6 adapter.
    • Teflon pipe thread tape can be used to hold the photodiode in the desired position.
  12. Mount each lens tube in the appropriate CP02 cage plates.
  13. Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.

Electronics subsystem

The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.

Parts

Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.

  • 1 electronic breadboard
  • 1 jump wire kit
  • 2 red 3' banana cable or similar
  • 1 black 3' banana cable or similar
  • individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench
  • 1 62 Ω resistor from the center counter top on the South wall
  • 1 15 kΩ resistor
  • other resistors, as-needed for the amplifier
  • 1 blue LED from the DNA Melting drawer at the far right of the wet bench
  • 2 LF411 op-amps from the electronics drawer
  • 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.

Assembly instructions

LED circuit

The led circuit consists of a 62 Ω resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 Ω resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.

  1. Build the LED circuit on your electronic breadboard.
    • Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.
  2. Disable the output of the lab power supply.
  3. Connect the LED circuit to the fixed 5 V output.

Temperature detection circuit

A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation RRTD=1000 Ω + 3.85 θ, where θ is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature.

The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 kΩ. Use a 15 kΩ resistor.

  1. Find an equation for Vout as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.
  2. Turn off the output of the triple-output power supply.
  3. Connect the RTD to the electronic breadboard.
  4. Measure the resistance of the 15 kΩ resistor with a DMM and record the value in your lab report.
  5. Connect the 15kΩ resistor in series with the RTD.
  6. Set the power supply for SERIES operation.
  7. Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.
  8. Set the current limit to 0.1 A.
  9. Connect the positive terminal of CH1 to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.
  10. Connect the negative terminal of CH2 to a banana terminal on the breadboard for -15 V.
  11. Connect the positive terminal of CH2 to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.
    • Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.
  12. Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 kΩ resistor.
  13. Turn on the output of the power supply and measure the voltage across the RTD using the DMM.
  14. What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?
  15. What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?
  16. How is the temperature calculation affected by a 1% change in the 15 V input voltage?

Photodiode amplifier circuit

The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.

Multi-stage photodiode amplification circuit

You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 106 Ω.

  1. Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.
  2. Construct the circuit on your breadboard.
    • Disable the power supply before you build.
    • Do not yet connect the photodiode.
    • Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.
  3. Connect the +/- 15 V bus strips to power the op-amps power pins.
  4. Connect the vertical blue bus strips to the horizontal blue bus strip.
Test connectivity and basic operation
  1. Verify that all your component connections are correct and match the design.
  2. Check that wires and components are properly seated in the breadboard and not suspiciously loose.
    • You can use the ohm-meter function of the digital multi-meter to verify continuity.
  3. Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.
  4. Disconnect the photodiode connection to pin 2 (the $ v^- $ input to the op-amp) and measure the output voltages of IC1 and IC2.
    • With the photodiode disconnected, the current signal is zero. If you see a significant offset voltage (from 50 to 100 mV) at IC1 pin 6, then you may wish to add (or adjust the value of) $ R_6 $.
    • Refer to Real Electronics, real op-amps or ask an instructor to understand and address this non-ideal behavior.

Data acquisition hardware subsystem

Each lab PC is equipped with a National Instruments USB-6212 or USB-6341 data acquisition (DAQ) card.

The USB-6212[1] has 16, 16-bit analog input channels which can, in sum total, accomplish 400 thousand samples per second (400kS/s). That is, if there are two channels, each one will be alternately sampled, and EACH sampled at 200kS/s. A multiplexer sequentially selects from among the 16 single-ended and 8 differential input signals. The card also supports two 16 bit analog output channels with an update rate of 250 kS/s and an output range of +/-10 V and up to +/-2 mA.

The USB-6212 also has 32 digital input/output channels and a digital ground. A 50 kΩ pull-down resistor is typically used in series with connections to these channels.

The USB-6341[2] is s little more powerful. It has 16, 16-bit analog input channels which can, in sum total, accomplish 500 thousand samples per second (500kS/s). Again, a multiplexer sequentially selects from among the 16 single-ended and 8 differential input signals. The card also supports two 16 bit analog output channels but they have an update rate of 900 kS/s and an output range of +/-10 V and up to +/-2 mA.

Finally, the USB-6341 has 24 digital input/output channels and a digital ground, as well as 4, 100 MHz counter/timers.

Summary of DAQ inputs/outputs

A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized in a memo.

Signal Name Signal Location Ground Location Pin wire color
DAQ Inputs
RTD AI0+ AI0- +Orange / -Black [lone pair, not with third (red) wire]
Photodiode AI1+ AI1- +Green / -White
DAQ Connection Cable

DNAMelter software

BasicDNAMelterIcon.png

Use the Basic DNA Melter GUI program (located on the lab computer desktop) to collect data from the apparatus.

At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the load command.

Documentation for the DNAMelter software is available here: DNA Melting: Using the Basic DNAMelter GUI

If you need to debug the DAQ (skip otherwise!)

  • When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.
    • Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.
    • In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."
    • If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in.
    • If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1."
    • Select the "Test Panels" tab to manually control or read signals from the DAQ.
  • If you run DNAMelter and there is a data acquisition error, for example, "Data acquisition cannot start!", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.
  • The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).
  • If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.

Part 1 measurements

Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.

Making samples


Biohazard.jpg LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under ../EHS Guidelines/MSDS Repository in the course locker for more information.


Sample prep steps

  1. Pipet 500 μL of DNA plus dye solution into a glass vial.
  2. Pipet up to 200 μL of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial.
    • Keep the sample vertical to make sure the oil stays on top.
  • You can use the same sample for several heating/cooling cycles.
  • Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low.

To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.

Sample disposal


Global Tree.gif Discard pipette tips with DNA sample residue in the pipette tips or the Biohazard Sharps container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.


Testing the instrument

Once your apparatus is built, use samples of fluorescein (~ 30μM concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.

Making a simple measurement of your instrument's low frequency SNR

Measure the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water.

  1. Run the Basic DNA Melter GUI.
  2. Place a vial of DI water in your instrument.
  3. Clear the data and wait 10 seconds.
  4. Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data.
    • Be sure that all other conditions, such as temperature, are stable throughout the test.

Compute the signal to noise ratio, $ \text{SNR}=\frac{\langle V_{fluorescein} \rangle - \langle V_{water} \rangle}{\sigma_{fluorescein} } $, where $ V_{fluorescein} $ is the portion of the data recorded with a fluorescein sample, $ V_{water} $ is the portion of the signal corresponding to the water sample, and $ \sigma_{fluorescein} $ is the standard deviation of $ V_{fluorescein} $.

Experiment steps

Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for a 20 bp DNA and green dye solution.

  1. Open and run the Basic DNA Melter GUI in Matlab and follow the instructions there.
  2. Confirm that block temperature is near room temperature when the Diablotek supply is off.
  3. Test a full heat/cool cycle with no DNA: Apply heat to the block by switching the Diablotek on to increase the block temperature to 95°C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.
    • Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.
  4. Now prepare a sample using the stock of "beater" (possibly labeled as "junk") DNA. Don't worry, it melts just as well as your fresh samples will melt.
  5. Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.
  6. Using the same temperature cycle protocol as in your test above, measure at least one melting curve of the beater DNA sample. Repeat as-desired to understand instrument operation.
    • Be sure to save a copy in a local directory so you can plot it in your Part 1 report.

Report requirements

  • One member of your group should submit a single PDF file to Stellar in advance of the deadline. The filename should consist of the last names of all group members, CamelCased, in alphabetical order, with a .pdf extension. Example: CrickFranklinWatson.pdf.
  • Include answers to questions embedded in the lab manual (e.g. the questions in the RTD section)
  • The file must be less than 20 MB.
  • Include code at the end of the document in an appendix, in the same pdf file, not as as separate upload.
  • Not counting the appendix, your report should be no longer than 10 pages.

Part 1 report outline

  1. Document your circuit design, optical design, and any ways that your instrument differs from the system described in the lab manual.
    • Include values for all of the resistors, capacitors in your circuit.
    • If you have not modified the circuits from their form in the report, you do not need to include the schematic in your report.
    • Draw a block digram of your optical system, including focal lengths of lenses and key distances.
    • Include a picture of your instrument.
  2. Report your signal to noise measurement.
  3. Plot at least one melting curve.
    • The plot should have temperature in °C on the horizontal axis and fraction of double stranded DNA on the vertical axis.
    • On the same set of axes, include a simulated curve generated by DINAMelt, OligoCalc, or another software simulator.
    • Also on the same set of axes, plot the output of the DnaFraction function evaluated with best-fit parameters. You may use nlinfit to choose the best-fit parameters or you may choose them manually. (See: DNA Melting: Simulating DNA Melting - Basics)
    • Include a legend.
  4. Report the estimated melting temperature and the best-fit values of ΔH°, ΔS°.
  5. Explain the statistical method you will use to identify your group's unknown sample in part 2 of this lab.

Lab manual sections

Lab manual sections

References

  1. Datasheet for the USB-6212
  2. Datasheet for the USB-6341

Subset of datasheets

(Many more can be found online or on the course share)

  1. National Instruments USB-6212 user manual
  2. National Instruments USB-6341 user manual
  3. Op-amp datasheet

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