20.309:Measuring DNA Melting Curves
Introduction
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In this lab, you will measure the melting temperature of several DNA samples to determine the effect of sequence length, ionic strength and complementarity. A common application of this technique exploits the length dependence of DNA melting temperatures to examine PCR products in order to determine whether a desired sequence was successfully amplified. The measurement technique utilizes a fluorescent dye that binds preferentially to double stranded DNA (dsDNA). This characteristic of the dye allows the relative concentration of dsDNA to be determined by measuring the intensity of fluorescent light given off by an excited sample. The DNA melting apparatus you will construct consists of four major subsystems: excitation, fluorescence measurement, temperature sensing, and data acquisition. You will build these subsystems out of an LED, a photodiode, a resistance temperature detector (RTD), and a PC data acquisition system.
The goal of your time in the lab will be to measure fluorescence intensity versus temperature for each of the samples over a range of about 90 degrees C to room temperature. This will provide a basis for estimating the melting temperature, Tm of each sample. (Tm is defined as the temperature where half of the DNA in the sample remains hybridized.)
Three of the samples will be unknown. All of them will have the same length, but different degrees of complementarity: complete match, single mismatch, and complete mismatch. Using the data you gather, you will attempt to identify these three samples.
Overview of the apparatus
In most DNA melting apparatuses, the temperature of the sample is ramped up at a controlled rate. In our homebrew setup, however, we will first heat up the sample on a hot plate. That way, natural cooling will provide the range of temperature conditions needed. As the sample cools, a PC data acquisition card will record the photodiode and RTD outputs over time. During data analysis, you will convert these voltages to temperature and relative dsDNA concentration. The melting temperature, Tm can be estimated from a graph of this data or its derivative.
When bound to dsDNA, SYBR Green I is most sensitive to blue light with a wavelength of 498 nm. The dye emits green light with a wavelength of 522 nm. You can easily observe this – a room-temperature sample of dsDNA and SYBR green looks yellow from the combination of blue excitation and green fluorescence. At higher temperatures, when there is no dsDNA to bind to, the sample will appear blue or clear.
In addition to dsDNA concentration, SYBR Green's fluorescence intensity dependencs on temperature. Higher temperatures reduce its fluorescence. This introduces an approximately linear error term into the signal as the temperature is ramped. The temperature dependence of SYBR Green can be accounted for in a variety of ways during analysis, including taking the derivative of the data.
As shown in the diagram, an aluminum heating block holds a cuvette containing the sample under test. The sample is a combination of DNA and a fluorescent dye called SYBR Green. In addition to being a convenient holder, the block gives the setup enough thermal inertia to facilitate a measurement from natural cooling. (Without the block, the sample would cool too quickly.)
Blue light from an LED illuminates one side of the cuvette. An optical filter shapes the output of the LED so that only the desired spectral range falls on the sample.
In the DNA melting apparatus, a photodiode placed at 90 degrees to the LED source detects the green light emitted by bound SYBR Green. The photodiode is placed behind an optical filter to ensure that only the fluorescent light given off by the sample is detected.
Since photodiodes produce only a very small amount of current, it will be necessary to build a very high gain transimpedence amplifier to produce a signal that is measurable by the PC data acquisition cards. Photodiode amplifiers are particularly challenging because many of the non-ideal characteristics of op amps become apparent at high gain.
An RTD attached to the heating block and wired to a voltage divider provides an indication of temperature. The temperature of the heating block will be a proxy for the sample temperature. Unfortunately, the block cools faster when it is hot than when it is near room temperature. You will have to get the heating block set up in your apparatus quickly after you remove it from the heating block.
The amplified photodiode output and the voltage across the RTD will be connected to two channels of the CP data acquisition card. A virtual instrument (called a VI) written in LabView will record the RTD and diode outputs. The DNA melting VI has a button to save data in file that can be loaded into Matlab for analysis.
Objectives and learning goals
- Measure temperature with an RTD.
- Implement a high gain transimpedence amplifier for photodiode current multiplication.
- Measure light intensity with a photodiode.
- Build an optical system for exciting the sample with blue light and gathering the fluorescence output on the photodiode.
- Record dsDNA concentration versus temperature curves for several samples.
- Estimate Tm from your data.
- Compare the measured DNA melting with theoretical models.
- Identify unknown DNA samples.
Lab procedure
Roadmap
- Build an optical system containing the LED, heating block, sample, photodiode, filters, and lenses.
- Hook up a three terminal voltage regulator to create an electrical power supply for the LED.
- Build, test, and calibrate the temperature-sensing circuit.
- Build an amplification/offset circuit for the DNA fluorescence signal.
- Troubleshoot and optimize your system.
- Heat a samples of DNA with SYBR Green dye and record DNA melting curves as the samples cool.
- Analyze the data. Identify a single-base mismatch (SNP) sequence. Compare your observations to theoretical models.
Optical system
TODO: Add figure with diagram of setup. TODO: Add picture of example setup.
The optical system consists of an LED, excitation filter, sample cuvette, heating block, emission filter, photodiode, optional lenses, and associated mounting hardware. Construct your system on an optical breadboard. The breadboard has a grid of tapped holes for mounting all kinds of optical and mechanical hardware. ThorLabs manufactures most of the hardware stocked in the lab. A few of the components you will certainly use include: 1/2" diameter posts, CP02 cage plates, and 1” diameter lens tubes.
Use optical rails and rail carriers or optical bases to mount 1/2” posts on the breadboard. RA90 right angle post clamps and post holderscan also be useful.
There are a variety of ways to construct your apparatus. A good design will be compact, stable, and simple. You will have to shield the optical system from ambient light, so a small footprint will be advantageous.
Illumination
Begin by mounting the LED on your breadboard. Note that there are two styles of LEDs. The Lamina LED Array is mounted on an aluminum heat sink and bolted to a CP02 cage plate. The CP02 attaches to the top of a post. It has an SM1 threaded hole through the middle that connects to 1” diameter lens tubes. The Cree LEDs are already mounted in a 1” lens tube.
Both styles of LED emit a range of wavelengths with a peak at 475 nm. A Chroma Technology D470 filter eliminates unwanted parts of the spectrum that might interfere with detection of the fluorescence signal. The filters have exposed, delicate coatings and must be handled carefully. In addition, the filter works better in one direction than the other.
Light from both kinds of LEDs diverges in a cone with an angle of about 100 degrees, so place the device close to the sample. You can also use a lens to concentrate the LED's output. Several lenses are available in the lab:
Photodiode
The photodiode is mounted in a short tube with SM05 threads. Use a SM1A6 adapter to mount the photodiode in a CP02 cage plate. Mount the photodiode assembly to the breadboard at 90 degrees to the LED. Build a system to hold the emission filter in front of the photodiode. You can use a lens to focus light from the sample on to the detector to improve performance, if you like.
Put an optical quick connect at the end of the photodiode assembly to facilitate easy attachment of the heating block during experimental runs. The other half of the quick connect goes into the CP02 cage plate mounted on the heating block.
Electrical System
Temperature
The electrical resistance of most materials varies with temperature. An RTD is a special resistor (usually made out of platinum) that exhibits a nearly linear change in its value with temperature. An RTD may be used to accurately measure temperature by including it as an element in a voltage divider. As the resistance of the RTD changes, so will the voltage across it.
A PPG101A1 RTD has been pre-mounted to the DNA heating block. This RTD has a nominal resistance value of 1 KΩ and its value increses with temperature. Note that the maximum current carrying capacity of this device is 1 ma. Hook up the RTD in a voltage divider. Make sure the divider has no more than 1 mA flowing through it. Use freeze spray or heat the block on the warmer to test the circuit.
Fluorescence intensity
Amplification circuit
Photodiode amplifiers can be fiddly under the best of circumstances. At such high gain, many of the non-ideal behaviors of op amps become apparent. It will be important to keep your wiring short and neat. Several other tips for building a good amplifier are given in the lab procedure section.
TODO: Insert Figure 3: Basic transimpedance amplifier circuit.
The photodiode (DATASHEET) you will use in to measure the fluorescence output produces only a tiny current – on the order of nanoamps. To measure and record the fluorescence signal, this signal must be amplified and converted to a voltage measurable by the PC data acquisition system. A transimpedance amplifier (sometimes called a current-to-voltage converter) with a gain of approximately 10<super>8</super> V/A will be required. Be sure you understand why this is the case, and explain it to your lab instructor.
The simplest transimpedance amplifier looks like Figure 3: Determine the DC gain Vout=iin of this configuration (in V/A) in terms of the resistance Rf . It should be clear that getting a large gain from this circuit requires the use of a very large resistor, which we'd prefer to avoid. (Optional: why not simply use a resistor, and omit the op-amp?)
Figure 4: The high-gain version of the transimpedance amplifier circuit. Don't forget to add a capacitor for high-frequency noise rejection.
Instead, we can use the circuit from Homework Set 1, which you have already analyzed, shown in Fig. 4. Since the signal you will measure will be very slowly-varying (near DC), you will also want to include a capacitor for rejecting high-frequency fluctuations (as discussed in HW1).
Note: as you build the setup, keep stability in mind. This is a sensitive high-gain
system, and it will not perform well if it is constantly getting bumped and wires are
being moved or disconnected. This means making solid electrical connections, keeping
wires and cables clamped or taped down, and setting things up to move as little as
possible when you connect and disconnect the sample. One final note about the type of op-amp to use. Though real op-amps don't behave exactly like ideal ones we use for analysis, we want an op-amp whose input current is as close to the ideal value of zero as possible (why?). Op-amps with this characteristic have JFET inputs, and in our lab, the LF411 or LF351 are both suitable.
Offset circuit
Figure 5: Offset circuit using the LM741. Note that all three of the pot's leads are used, and the wiper is connected to the negative sup- ply voltage.
A final addition to the system that will greatly improve its usabil- ity is a knob that lets you control the level of the output signal. A simple way to do this is using an LM741 op-amp, with a 10k potentiometer connected as shown in Fig. 5. Use one of the high- quality round pots to obtain smooth and precise control.
TODO: Figure 6: Recommended layout of the light source, filters, and detector for the optics setup.
Optical system
The LED array connects to the circuit just like a single LED, using two leads - anode and cathode. It can be powered directly from your power supply, and ¼ 8:8V is known to supply a good amount of light while dissipating manageable heat.
PC Data Acquisition System
TODO: describe the DAQ system and capabilities.
Sample handling
The DNA samples for these experiments are loaded inside a glass cuvette. You should use 500¹L of DNA solution for each run. Pipet (20¹L) of mineral oil on top of the DNA solution to prevent evaporation; make sure the oil stays on top by always keeping the block vertical, especially when heating it.
To change samples, pipette out the previous DNA solution, and discard it in the waste container provided. Discard the pipette tip. Rinse the cuvette with water, then, using a fresh pipette tip, fill it with 500¹L of new DNA solution. You should be able to use the same DNA sample for many heating/cooling cycles, so only replace it if you lose significant volume due to evaporation. If you need to leave the sample overnight, store it in the lab refrigerator, and clearly label it with your name.
Heating block
For better temperature stability and easier handling, the cuvette slips inside a machined aluminum block, also provided for you. The RTD RTD for temperature measurement is hard-mounted onto this block. Use a hotplate to heat up the block to above the DNA melting temperature (¼ 90ffC), then move the block to your setup for measurement, and connect the RTD to the Wheatstone circuit.
You'll find that the speed at which the block cools down is likely too fast if it's placed directly in contact with the metal optical breadboard. You can control this speed by placing one or a few pieces of paper or layers of paper towel underneath the block. Experiment with this until the block takes between 5 and 10 minutes to cool down from 90ffC to 40ffC.
Experimental Protocol
Preparing the setup
- The system will generate the best data when both the amplifier circuit and LED have been on for at least 60 min. and all drifts have stabilized. However, you certainly don't need to wait idly while this happens, and can test the setup and try measurements in the meantime.
- Do this first without the sample and the block. Using a box and a piece of black cloth, make sure the entire optical setup is isolated from stray light. If you can see any blue light coming out at all, light is also getting in.
- Use the potentiometer to adjust the amplifier voltage offset until the baseline signal is approx. 0 V.
- The baseline should be relatively flat { if it is not, generally it's because the setup is not well covered.
- Now include the block with DNA solution in the setup. Adjust the block's placement and the angle of the LED source to maximize the ratio of DNA signal vs. background signal without
the block. Use the plastic alignment jig if you need, or simply mark the position of the block in some way.
DNA melting curve experiment
- Place the block on the hotplate (set to 95ffC) for at least 10 min. Measure the RTD RTD to make sure the temperature is above 80ffC before removing the block
- Move the block to your setup. Immediately connect the RTD to its Wheatstone bridge circuit. Cover the setup in a box and put a piece of black cloth over it.
- Start the LabVIEW recording program (called DNAmelting.vi). Wait for the block to cool to below 40ffC, which should take about 10 min. if the block sits on a single piece of paper towel.
- A monotonically decreasing fluorescence signal is expected (for an inverting amplifier setup). If there is any LED output fluctuation, or if the signal drifts upward, you'll need to restart the experiment. When you're satisfied with the melting run, save the data.
Report Requirements
Data Analysis
Once your instrument is running to your satisfaction, you should record the following melting curves:
- 40bp perfect match
- 19bp perfect match
- 19bp complete mismatch
- 19bp single-base mismatch (SNP)
- 19bp at 1-2 different ionic strengths
(Note: Other than the last one, the 19bp samples will only be identified as A, B, and C, and you will need to identify, based on your measurements, which is which.) You will need to take the derivative of the recorded fluorescence data, and combine it with the temperature data to generate plots. Generally, the region of interest will fall between 40 and 65ffC. It will be helpful to create a matlab script to convert raw data to a plot of dF/dT vs. temperature.
he \melting temperature" Tm is defined as the temperature at which 50% of the DNA remains hybridized. Sometimes, the transition is not particularly sharp, or other factors in the measurement may create offsets or drifts in the signal (evident below 80±C in Fig.1(a)), in which case the derivative of this curve is plotted (Fig. 1(b)), and the location of its peak value gives Tm more clearly. More about this in Section 4.2.3.
Having generated your melting profiles, you need to produce the following plots:
- Comparison of perfect match 40bp and 19bp sequences.
- Comparison of 19bp perfect match vs. SNP vs. complete mismatch sequences.
- Comparison of 19bp sequences at different ionic strengths.
In each situation, discuss the melting temperatures and shapes of the melting curves for the samples relative to each other. Briefly explain the curves are the way they are in each case. Compare your melting curves with those of other students in the class. You may find the quite different even under the same conditions. What might cause these variations? What factors affect the DNA melting temperature, and the \sharpness" of the melting transition?
Model vs. reality
In class, we derived an expression that relates the melting temperature to the enthalpy change ¢Hff and entropy change ¢Sff of the hybridization reaction:
T(f) =
¢Hff
¢Sff ¡ Rln(2f=CT (1 ¡ f)2) ; (1)
Here, f is the fraction of DNA strands hybridized (dimerized) at a particular temperature (at Tm, this is 1/2), and CT is the total concentration of single-strand oligonucleotides (or 2£ the dsDNA concentration when all strands are hybridized). Choose one of the perfect-match sequences that you measured, and use matlab to fit the model to your measured data, which will allow you to extract the ¢Hff and ¢Sff parameters. To perform the fit, you will need a matlab function that will evaluate T(f) given an input const for the ¢Hff and ¢Sff parameters. The function will be something like this:
function Tf = melt(const, f)
R=8.3;
C_T=33e-6;
dH = const(1);
dS = const(2);
Tf = dH./(dS - R*log(2*f./(C_T*(1-f).^2)));
You can then invoke matlab's lsqcurvefit routine to do the fit, which will return the best values for ¢Hff and ¢Sff.
FitVals = lsqcurvefit(@melt, [dH_guess, dS_guess], frac_vector, temp_vector)
Bonus (optional):
1. Calculate ¢Hff and ¢Sff for this sequence using the nearest-neighbor model from class.
2. Compare these to the fit parameters, and speculate about why they might be different? What
factors affect ¢Hff and ¢Sff?
External references
TODO: Bypass caps, grounding, don’t let diode shield touch ground
