20.309:Measuring DNA Melting Curves

Measuring DNA Melting Curves

Introduction

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 DNA melting apparatus you will construct consists of four major subsystems: excitation, temperature measurement, fluorescence measurement, and data acquisition.

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. Light intensity will be measured by a photodiode, which produces a small amount of electric current in proportion to the amount of light striking it. Temperature will be measured with a special kind of resistor called a thermistor. The goal of your time in the lab will be to produce graphs of fluorescence intensity versus temperature for each of the samples. These graphs provide enough information to estimate of the melting temperature of each sample.

Three of the samples will have the same length but different degrees of complementarity: complete match, single mismatch, and complete mismatch. Using the data you gathered, 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 samples needed. As the sample cools, a PC data acquisition card will record the photodiode and thermistor outputs over time. During data analysis, you will convert these voltages to temperature and relative dsDNA concentration. The melting temperature, T_m can be estimated from a graph of this 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. A thermistor 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. (It is possible that the sample’s temperature will differ from the block’s.)

Blue light from an LED illuminates one side of the cuvette. Under this illumination, the dye gives off green light when bound to dsDNA. You can easily observe this – the room-temperature sample will appear yellow from the combination of blue excitation and green fluorescence. At higher temperatures – when the dye is inactive – the sample will look blue or clear. A photodiode placed at 90 degrees to the illumination source detects light given off by the dye.

When bound to dsDNA, SYBR Green is most sensitive to blue light with a wavelength of 498 nm. The dye emits green light with a wavelength of 522 nm. The right angle placement of the photodiode prevents much of the direct illumination from reaching the detector. In addition, an optical filter placed after the LED ensures that only blue light in the desired range falls the sample. A filter in front of the photodiode selects only the green light emitted by the dye.

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.

Objectives and Learning Goals

-Measure temperature with a thermistor.

-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 T_m from your data.

-Compare the measured DNA melting with theoretical models.

Lab Procedure

Roadmap and Milestones

1. Hook up a three terminal voltage regulator to create a supply for the LED.
2. Build, test, and calibrate the temperature-sensing circuit.
3. Build an amplification/offset circuit for the DNA fluorescence signal.
4. Assemble an optics setup that will enable you to observe the light output of a DNA sample to be studied.
5. Troubleshoot and optimize your system.
6. Heat a samples of DNA and fluorescent dye ang record DNA melting curves as the samples cool.
7. Analyze the data. Identify a single-base mismatch (SNP) sequence. Compare your observations to theoretical models.

PC Data Acquisition System

TODO: describe the DAQ system and capabilities.

Illumination

There are two types of LEDs available in the lab. Here are links to the datasheets for the [LED Array] and the [LED].

As can be seen from on (at least the Cree) datasheet, the blue LED has a peak emission around 475 nm; however, there is nonzero light output at 522 nm. As such, a filter from Chroma Technologies will be used to

SYBR Green

Temperature sensing

A thermistor is a resistor whose resistance varies with temperature. Thermistors are available with positive and negative temperature coefficients (PTC and NTC). As apparent from the name, PTC thermistors have increasing resistance with higher temperature. An NTC thermistor decreases resistance with temperature. To build and test your temperature-sensing circuit, there are 5Kω NTC thermistor available. (Black with red leads, part number RL0503-2890-95-MS). As shown in the datasheet, these cheap-o thermistors have a nonlinear temperature-resistance characteristic. In the DNA melting apparatus, you will use a higher quality thermistor called a resistance temperature device (RTD). The RTD has a positive temperature coefficient and a nominal value of 1 Kω with very linear temperature response. Here is a link to the datasheet for the PPG102A1.

Wheatstone bridge

Though simple, the voltage divider is a very powerful concept in electronics { divider-based circuits contribute to many types of sensor and measurement systems. Here we will use a well-known circuit called a Wheatstone bridge to make temperature measurements. The Wheatstone bridge was invented in the 1800's in order to measure the resistance of an unknown value Rx. The circuit consists of four resistors in the configuration shown in Figure 2. Typically, two have known values, R1 and R2, and a third resistor R3 is variable. The unknown resistance Rx takes the fourth position, and the circuit is wired with a voltage source Vin. The bridge is \balanced" when the ratio of R1 to R3 is exactly equal to the ratio of R2 to R4, in which case the voltages at nodes a and b are equal. Otherwise a voltage difference develops, and current will flow if a connection is made across the bridge. Wire up a Wheatstone bridge on your breadboard, as shown in Figure 2, using a 1-10 k­potentiometer for R3 (blue rectangular, not the round multiturn type) and values for R1 and R2 Figure 2: A Wheatstone bridge circuit. that will give good sensitivity if Rx is in the 1 k­ range. Note that the RTD should have no more than 1 mA of current passed through it, so build your circuit accordingly. One way to measure the unknown resistance Rx is by monitoring the voltage difference Va ¡ Vb, and adjusting the value of R3 until the bridge is balanced. Another method is to perform a calibration to derive the relationship between the voltage across the bridge to the change in resistance. Our approach will be a combination of these methods { you will first adjust R3 to balance the bridge, then use your knowledge of how Vab depends on Rx to calculate resistances from voltages.

Test your temperature circuit using the black-with-red-leads thermistor, and small amounts of cold or warm water to vary its temperature. Observe the circuit output and make sure that the temperature reading makes sense to you. A thermometer is likely to be helpful here. Once you are satisfied with this, connect your circuit to a the sample block with the attached RTD. Make sure you are able to convert Vab to resistance, and use the datasheet to determine the calibration relationship between temperature and resistance. You should now have a functional electronic thermometer, and a straightforward way to convert its output to temperature.

Fluorescence readout system

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.

SYBR Green

This is a common intercalating dye that binds highly preferentially to dsDNA (not ssDNA), and exhibits strong fluorescence when bound and nearly zero fluorescence when unbound. We therefore expect to see an decreasing signal (using an inverting configuration) as the temperature of the DNA sample drops, and more and more DNA duplexes are formed. SYBR Green I is ex- cited by light at a wavelength of 498 nm (blue), and emits at 520 nm (green). SYBR Green I fluorescence is not only dependent on its bind- ing state with dsDNA, but also has a dependence on temperature. Higher temperatures reduce its fluorescence, which introduces an approximately-linear drift into the signal as the temperature is ramped. This is one of the reasons for taking the derivative of the recorded data to determine Tm.

TODO: Figure 6: Recommended layout of the light source, filters, and detector for the optics setup.

Optical system

Figure 6 shows a schematic of the recommended optical configuration. The right-angle geometry helps to reduce any background signal that gets through the excitation filter. Build this on an optical breadboard, keeping the components close to the board for stability and simplicity of design. This will mostly involve short rails, rail clamps, and posts. The filters used are a D470/40x bandpass filter (excitation), and a E515LPv2 long-pass filter (emission) from Chroma Technologies. Take care not to touch the optical surfaces when handling these components, as they have coatings that are easily damaged. Datasheets are available, as usual.

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.

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 thermistor 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 thermistor 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

1. 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.

1. 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.

1. Use the potentiometer to adjust the amplifier voltage offset until the baseline signal is approx. 0 V.

1. The baseline should be relatively flat { if it is not, generally it's because the setup is not well covered.

1. 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

1. Place the block on the hotplate (set to 95ffC) for at least 10 min. Measure the RTD thermistor to make sure the temperature is above 80ffC before removing the block
2. 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.
3. 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.
4. 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:

1. 40bp perfect match
2. 19bp perfect match
3. 19bp complete mismatch
4. 19bp single-base mismatch (SNP)
5. 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:

1. Comparison of perfect match 40bp and 19bp sequences.
2. Comparison of 19bp perfect match vs. SNP vs. complete mismatch sequences.
3. 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?

TODO: Bypass caps, grounding, don’t let diode shield touch ground