|Temperature cycling fluorometer apparatus.
||Example DNA melting curves showing the effect of varying ionic strength.
Now that you've refreshed your memory on the basics of electronics, you'll apply them by building an instrument capable of measuring DNA melting and DNA annealing.
What exactly do we mean by that?
Annealing reaction of two complementary olives. See the this wiki page
for a review of the temperature-dependent equilibrium between single-stranded and double-stranded DNA in solution. Several web-based tools are available to predict melting melting curves, including DINAMelt Web Server
Short strands of DNA from about 10-50 base pairs in length are called "oligonucleotides" or just "oligos." In solution, DNA oligos exist in a temperature-dependent equilibrium that consists predominantly of double-stranded helicies and single-stranded random coils. The forward reaction in which two complementary, single stranded DNA (ssDNA) molecules combine to form double-stranded DNA (dsDNA) is called annealing. The reverse process is called thermal denaturation or melting. You can learn a lot about DNA oligos by melting them.
Double-stranded DNA predominates at low temperatures. As temperature increases, the equilibrium shifts from dsDNA to ssDNA. The equilibrium concentrations of single- and double-stranded DNA depend on several characteristics of the oligos and the solution, including the length of the oligos; the concentration of salt ions in solution; the percentage of AT versus GC base pairs; and the degree of complementarity. The reaction can be characterized by a DNA melting curve, which is a plot of dsDNA concentration versus temperature. DNA melting curves are the basis of several experimental and clinical techniques. High-resolution melting (HRM) analysis uses melting curves to measure features of DNA oligos such as differences in sequence, purity of PCR products, or even methylation state.
Fluorescent dyes with a quantum efficiency that dramatically increases when they are bound to dsDNA are available, such as SYBR Green and ResoLight. These dyes glow about 1000x more intensely when they are bound to dsDNA. In this lab, you will build an instrument called a temperature-cycling fluorometer that measures the change in fluorescence over a range of temperatures to generate melting curves for several DNA oligos. From the melting curves you measure, you will estimate the thermodynamic parameters of the DNA annealing reaction: ΔH°, ΔS° and the melting temperature. You will use your instrument to examine the effect of varying oligo length, ion concentration, or degree of complementarity on the thermodynamic parameters and to identify an unknown sample.
If you haven't studied DNA thermodynamics before (or if it has been a while since you have), see this review of DNA Melting Thermodynamics before you go on.
Raw data from the instrument consists of two voltages: one proportional to fluorescence, and another that depends on temperature. You will digitize the voltages with a computer data acquisition system and process the raw signals to produce DNA melting curves. The instrument has five major subsystems:
DNA melting apparatus block diagram.
Current flowing through a Peltier device
, also called a thermoelectric cooler (TEC), causes heat to flow from one surface of the device to the other. The direction of heat flow depends on the direction of the current. The DNA melter utilizes a Peltier device to heat and cool the sample.
- The sample consists of 30 μM complementary DNA oligos in solution with NaCl and a fluorescent dye.
- The absorption peak of the dye is 497 nm and the emission peak is 520 nm.
- Optical excitation
- A blue LED excites the sample.
- The LED produces a broad spectrum of light with a peak intensity around 475 nm.
- A band-pass optical filter eliminates wavelengths below 450 and above 490 nm.
- Temperature measurement and control
- The sample container is placed in an aluminum heating block to control its temperature.
- The heating block has holes drilled in it to allow optical access to the sample while it is being heated.
- Electric current drives a Peltier device to pumps heat in to or out of the heating block, depending on the direction flow.
- Temperature measured by a temperature-sensitive resistor called a resistance temperature detector (RTD) embedded in the heating block.
- Fluorescence detection
- A photodiode placed at 90° to the LED source detects light emitted by the fluorescent dye in the sample.
- A long-pass emission filter with a cutoff of 515 nm eliminates light from the LED.
- Since the photodiode produces a very small amount of current, you will construct a high-gain amplifier to provide a measurable voltage.
- Data Acquisition
- Data will be recorded by a computer DAQ (data acquisition) system.
- A program is provided to record these fluorescence and temperature signals over time and save the data to a file.
How to do this lab (assignments 7 through 10)
- Refresh your understanding of DNA Melting Thermodynamics.
- Follow the guidelines in Assignment 7 to build a system for exciting, heating, measuring fluorescence, and measuring temperature of a DNA sample.
- Troubleshoot and optimize your instrument.
- Measure the signal to noise ratio.
- Generate melting curves for a known sample.
- Estimate the melting temperature of the known sample. Turn in Assignment 7.
- Improve your instrument by adding temperature control and lock-in signal processing, as outlined in Assignment 8.
- Complete Assignment 9 where you will write the code to simulate and analyze DNA melting data using multi-parameter, nonlinear regression.
- Verify the performance of your instrument with known samples.
- Measure the melting curves of your three known and one unknown samples.
- Use your analysis code to extract even better estimates of ΔH°, and ΔS°, and use this refined knowledge to analyze your data. Your goal is to identify the unknown sample.
- Turn in [[Assignment 10 Overview| Assignment 10]. You'll have learned a lot by then!
Objectives and learning goals
- Build an optical system for exciting the sample with blue light and gathering the fluorescence output on the photodiode.
- Measure light intensity with a photodiode.
- Build a heating system to reliably heat and cool your sample.
- Measure temperature with an RTD and an appropriate transfer function.
- Implement a high gain transimpedance amplifier.
- Use a lock-in amplifier to reduce noise.
- Record dsDNA concentration versus temperature curves for several samples.
- Analyze the data to find the dsDNA fraction as a function of temperature.
- Estimate Tm from your data.
- Compare the measured curves with theoretical models.
- Identify unknown DNA samples.
Let's dive into it!
This assignment has 3 parts:
- Part 1: Solve problems by applying the 'golden rules' of ideal op amps;
- Part 2: Build an instrument to measure temperature and fluorescence.
- Part 3: Test your instrument and measure a preliminary melting curve.
Submit your work on Stellar in a single PDF file with the naming convention <Lastname><Firstname>Assignment1.pdf.
||Here is a checklist of all things you have to turn in:
- answers to all questions in Assignment 7, Part 1;
- your chosen values for resistors R1 through R5, and capacitor C1 in the transimpedance amplifier circuit.
- Calculate the resulting cutoff frequency for Stage 1, and the gain of Stage 1 and Stage 2 of your circuit.
- If you have not modified the circuits from their form on the wiki, you do not need to include the schematic in your report.
- your RTD calculations and measurements:
- Find an equation for the block temperature as a function of the voltage across the RTD.
- Measure and record the voltage across the RTD, as well as the source voltage of your circuit at room temperature.
- Calculate the temperature based on your measured voltages. How does it compare with the clock on the wall (or some better measure)?
- Assume your source voltage is 15V exactly. How is the temperature calculation affected by a 1% change in the 15 V input voltage? In other words, if you assume the same measured voltage across the RTD, but have a 1% change in the input voltage, how much does your calculated temperature change? Note the importance of keeping track of your input voltage.
- a detailed description of your DNA melting lab optical design, and any ways that your instrument differs from the system described in the lab manual;
- Draw a block diagram of your optical system, including focal lengths of lenses and key distances.
- your signal-to-noise measurement;
- 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 your simulated curve from the pre-lab problem B,2 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.
- Report the estimated melting temperature and the best-fit values of ΔH°, ΔS°.
Back to 20.309 Main Page
Additional suggested readings and references
Zipper H, Brunner H, Bernhagen J, Vitzthum F. Investigations on DNA Intercalation and Surface Binding by SYBR Green I, its Structure Determination and Methodological Implications. Nucleic Acids Res., 2004, 32(12), e103.
Hua-Xi Xu, Yoshiaki Kawamura, Na Li, Licheng Zhao, Tie-Min Li, Zhi-Yu Li, Shinei Shu and Takayuki Ezaki, A rapid method for determining the GMC content of bacterial chromosomes by monitoring fluorescence intensity during DNA denaturation in a capillary tube Int. J. of Sys. and Evo. Microbiolog, 2000, 34, 1463–1469.
Haukur Gudnason, Martin Dufva1, D.D. Bang and Anders Wolf, Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature Nucleic Acids Research, 2007, Vol. 35, No. 19 e127.
A more complete DNA melting and PCR resource list is available on this wiki. Please improve the page by adding relevant, high-quality sources.
- ↑ Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997;245: 154–60.
- ↑ Breslauer et al., Predicting DNA duplex stability from the base sequence PNAS 83: 3746, 1986
- ↑