20.309:Measuring DNA Melting Curves

Introduction

This is a work in progress. Please check back for the final version.

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, T_m of each sample. (T_m 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, T_m can be estimated from a graph of this data or its derivative.

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.

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. You can easily observe this – a room-temperature sample of dsDNA and SYBR green 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 LED source detects the green light. 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 T_m from your data.
• Compare the measured DNA melting with theoretical models.
• Identify unknown DNA samples.

Lab procedure

Roadmap

1. Build an optical system containing the LED, heating block, sample, photodiode, filters, and lenses.
2. Hook up a three terminal voltage regulator to create an electrical power supply for the LED.
3. Build, test, and calibrate the temperature-sensing circuit.
4. Build an amplification/offset circuit for the DNA fluorescence signal.
5. Troubleshoot and optimize your system.
6. Heat a samples of DNA with SYBR Green dye and 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.

Optical system

TODO: Add figure with diagram of 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 breadboards. The breadboard has a grid of tapped holes for mounting all kinds of hardware. ThorLabs manufactures most of the optical hardware stocked in the lab. The major hardware components of the DNA melting system are made from: 1/2" diameter posts, CP02 cage plates, and 1” diameter lens tubes.

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.

• Use optical rails and rail carriers or optical bases to attach φ1/2” posts to the breadboard.
• You can use a 1/4-20 screw directly in the bottom of a post or use a post holder. The post holder makes it much easier to adjust the height and orientation of components; however, directly mounted posts are a bit more stable.

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 cage plate has an 8-32 tapped hole on the bottom that attaches to a post and an SM1 threaded hole through the middle that connects directly 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.

In either case, build a system to hold the LED and excitation filter.

The coating on the [excitation filter are very delicate. Handle them by their http://web.mit.edu/~20.309/Labs/Datasheets/Chroma%20Filter%20Information.pdf

Light leaving the LED diverges in a cone with an angle of about 100 degrees. You can improve the performance of the illuminator by using a lens to concentrate the output of the LED on the sample. Several lenses are available in the lab:

• f=25.4mm
• f=50mm
• f=100mm

*f=200mm

TODO: add handling of filter

Photodiode

The photodiode has 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. Mount 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.

Put an optical quick connect on your setup to facilitate quick attachment of the heating block. (Put the other end of the quick connect into the CP02 cage plate mounted on the heating block.)

Wiring up the LED and photodiode

Todo: outline wiring, describe voltage regulator.

Temperature sensing

An RTD is a resistor (usually made out of platinum) with a resistance value that varies with temperature. This useful property of RTDs may be exploited by including them in as an element in a voltage divider. As the resistance of the RTD changes, so will the voltage across it. The PPG101A1 RTD we will use in the lab has a

Hook up the RTD in a voltage divider. The divider should have no more than 2 mA flowing through it. (The power supply will be +15V.)

To build and test your temperature-sensing circuit, there are 5Kω NTC RTD available. (Black with red leads, part number RL0503-2890-95-MS). As shown in the [DATASHEET|datasheet], these cheap-o RTDs have a nonlinear temperature-resistance characteristic. In the DNA melting apparatus, you will use a higher quality RTD 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 [DATASHEET NEEDED|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 RTD, 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

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

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 RTD 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?

External references

• National Instruments data acquisition card user manual

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