1 20.309: Biological Instrumentation andMeasurement Laboratory Fall 2006 Module 1: Measuring DNA Melting Curves Contents 1 Objectives and Learning Goals 1 2 Roadmap and Milestones 2 3 Background 2 4 Building the apparatus 3 4.1 Temperature sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4.1.1 Thermistor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4.1.2 Wheatstone bridge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4.2 Fluorescence readout system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4.2.1 Amplification circuit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4.2.2 Offset circuit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4.2.3 SYBR Green I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4.2.4 Optical system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.2.5 Sample handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.2.6 Heating block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5 Experimental Protocol 7 5.1 Preparing the setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5.2 DNA melting curve experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 6 Report Requirements 8 6.1 Data to take . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 6.2 Model vs. reality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Objectives and Learning Goals Implement a temperature sensor using the well-known Wheatstone bridge scheme. • Build an instrument for recording DNA melting curves. • Use your instrument to study the melting behavior of several DNA samples under different • conditions. Understand the factors affecting DNA melting behavior, and what type of information can • be usefully obtained from such measurements. 120.309: Biological Instrumentation andMeasurement Laboratory Fall 2006 2 Roadmap and Milestones 1. Build and test the temperature-sensing circuit. 2. Calibrate the circuit for accurate temperature measurement. 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. Combine the subsystems you have built to generate a DNA melting curve; troubleshoot and optimize your system. 6. Obtain DNA melting data for several sequences, and identify a single-base mismatch (SNP) sequence. 3 Background You will learn in lecture about the im-portance and utility of measuring of DNA melting temperatures. A com-mon application takes advantage of the length-dependence of DNA melt-ing temperatures to examine PCR prod-ucts, and determine whether the desired sequence was successfully amplified. The goal of this lab is to build the hardware to be used for double-stranded DNA (dsDNA) concentration measure-ment with the common dsDNA-binding dye SYBR Green I. This setup, together with a temperature sensing circuit, en-ables DNA length and complementar-ity analysis via melting curve measure-ment. The tools developed in this lab will also be capable of analyzing other fluorescent dyes with similar excitation and emission wavelengths. A typical plot of temperature-dependent fluorescence from a solution of DNA and SYBR Green I is shown in Fig. 1. The “melting temperature” Tm is defined as the temperature at which 50% of the DNA remains hybridized. Sometimes, the transition is not partic-ularly sharp, or other factors in the mea-surement may create offsets or drifts in the signal (evident below 80◦C in Fig. Figure 1: Typical melting curves using SYBR Green I. 1(a)), in which case the derivative of this 2 0.350.30.250.20.150.10.05060 65 70 75 80 85 90Temperature (deg C)dRFU/dT6543210-160 65 70 75 80 85 90Temperature (deg C)RFU(a)(b)Figure by MIT OCW.20.309: Biological Instrumentation andMeasurement Laboratory Fall 2006 curve is plotted (Fig. 1(b)), and the lo- cation of its peak value gives Tm more clearly. More about this in Section 4.2.3. To perform a DNA melting experiment, the temperature of the DNA sample is usually ramped up at a controlled rate, and carefully monitored, while the concentration of dsDNA is recorded, most often using a fluorescent dye. To avoid the need for precise temperature control, we will ramp the temperature downward by heating the DNA sample to ab ove its melting temperature, and letting it cool naturally. 4 Building the apparatus Your apparatus will consist of two major subsystems for (a) temperature measurement, and (b) quantification of dsDNA in your sample. The outputs of the subsystems give you the two data sets that constitute a DNA melting curve: temperature and relative fluorescence intensity. 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. 4.1 Temperature sensing 4.1.1 Thermistor A thermistor is a resistor whose resistance varies with temperature. Thermistors come in two “fla-vors:” positive temperature coefficient (PTC – R increases with higher T) and negative temperature coefficients (NTC – R decreases with higher …