Difference between revisions of "Lab Manual: Measuring DNA Melting Curves"

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[[Image:Example Melting Curve.jpg|thumb|right|250px|Example DNA melting curves showing the effect of varying ionic strength. The data has been filtered to reduce noise.]]
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{| class="wikitable" style="margin: 1em auto 1em auto;"
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[[Image:Example Melting Curve Derivative.jpg|thumb|right|250px|Differentiating the melting curve simplifies finding T<sub>m</sub>.]]
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| width="325px" |[[Image:DNA_Excitation.JPG|300px]]
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| width="325px" |[[Image:Example Melting Curve.jpg|300px|]]
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|-
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| width="325px" |Temperature cycling fluorometer apparatus.
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| width="325px" |Example DNA melting curves showing the effect of varying ionic strength.
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==Overview==
 
==Overview==
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In this lab, you will build a temperature cycling fluorometer &mdash; an instrument that measures fluorescence intensity as a function of sample temperature. You will use the instrument you build to measure solutions of DNA oligos, salt ions, and a fluorescent reporter that binds to double stranded DNA (dsDNA). The instrument will produce two signals: one proportional to fluorescence, and another that depends on temperature. You will process the raw data to produce a plot of dsDNA fraction versus temperature, called a DNA melting curve. From the melting curve, you will estimate the thermodynamic parameters of the DNA annealing reaction: ''&Delta;H&deg;'', ''&Delta;S&deg;'' and the melting temperature of the oligo.
+
[[Image:DNA_strands_in_solution.gif|thumb|right|300 px|Annealing reaction of two complementary olives. See the  [[DNA Melting Thermodynamics|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 [http://mfold.rna.albany.edu/?q=DINAMelt/Hybrid2 DINAMelt Web Server] and [http://www.basic.northwestern.edu/biotools/OligoCalc.html Oligocalc]. ]]
 +
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.<ref>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.</ref>
  
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The thermodynamic parameters depend on several factors, including the length of the oligos; concentration of salt ions; AT versus GC content; and the degree of complementarity of the oligos. A review of DNA melting thermodynamics is available on [[DNA Melting Thermodynamics|this page]].
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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.<ref>Breslauer et al., [http://www.pnas.org/content/83/11/3746.full.pdf Predicting DNA duplex stability from the base sequence] PNAS 83: 3746, 1986</ref> 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.<ref>[https://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_070933.pdf ]</ref>
  
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[[Image:DNA_Excitation.JPG|thumb|right|350px|DNA Melting Apparatus]]
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Fluorescent dyes with a quantum efficiency that dramatically increases when they are bound to dsDNA are available, such as [http://en.wikipedia.org/wiki/SYBR_Green_I SYBR Green] and [https://lifescience.roche.com/shop/products/lightcycler14301-480-high-resolution-melting-dye 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: ''&Delta;H&deg;'', ''&Delta;S&deg;'' 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.
−
The instrument has five main subsystems:
+
  
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* optical excitation
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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.
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* temperature control
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* fluorescence detection
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* temperature measurement
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* data acquisition and control
+
  
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A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.
+
==Instrument==
 +
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:
  
−
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.
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[[Image:DNA Melting Block Diagram.jpg|thumb|350 px|DNA melting apparatus block diagram.]]
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[[Image:TEC_picture.jpg|thumb|right|Current flowing through a [https://en.wikipedia.org/wiki/Thermoelectric_cooling 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.]]
  
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The sample block will be heated by a TEC and its temperature measured by an embedded RTD. Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T and from a comparison to the thermodynamic model.
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* Sample
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The thermodynamic parameters of DNA melting depend on the sequence length, salt ion concentration, and degree of complementary between the two oligos. 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 emitted by an excited sample.  
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** The sample consists of 30 &mu;M complementary DNA oligos in solution with NaCl and a fluorescent dye.
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** The absorption peak of the dye is 497 nm and the emission peak is 520 nm.
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You will measure samples of both known and unknown composition. The samples may vary in length, complementarity (complete match, single mismatch, or complete mismatch), or salt concentration. You will compare the data you gather to a theoretical model and you will attempt to identify unknown samples.
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* Optical excitation
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** A blue LED excites the sample.
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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.
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** 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&deg; 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==
 
==How to do this lab==
  
−
#Refresh your understanding of [[DNA Melting Thermodynamics]]
+
[[Image:DNA Melting Lab Part 1.png|700 px]]
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#Complete the Simulating DNA Melting homework using [[DNA Melting: Simulating DNA Melting - Basics]] and learn more about the signals you will observe in the lab and how to start to analyze them.
+
−
#Follow the guidelines in [[DNA Melting Part 1: Measuring Temperature and Fluorescence|Part 1]] of the lab to build a system for exciting, heating, measuring fluorescence, and measuring temperature of a DNA sample.
+
−
#Troubleshoot and optimize your instrument, and measure the signal to noise ratio.
+
−
#Generate melting curves for a known sample.
+
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#Estimate the melting temperature of the known sample. Turn in [[DNA Melting Report Requirements for Part 1|Part 1 of your lab report]].
+
−
#Improve your understanding of noise sources and other non-idealities by completing additional simulation in [[DNA Melting: Simulating DNA Melting - Intermediate Topics]].
+
−
#Improve your instrument by adding lock-in signal processing and temperature control, as outlined in [[DNA Melting Part 2: Lock-in Amplifier and Temperature Control|Part 2]] of the lab.
+
−
#Verify the performance of your instrument with the known sample.
+
−
#Measure your known and unknown samples.
+
−
#Attend the tutorial on multi-parameter, nonlinear regression to estimate ''&Delta;H&deg;'', and ''&Delta;S&deg;'', and use to analyze your data from Part 2.
+
−
#Turn in [[DNA Melting Report Requirements for Part 2|Part 2 of your lab report]]. In this final report submission, include Parts 1 and 2 and note any significant revisions that you may have made to Part 1 due to your increased understanding.
+
−
 
+
  
 +
# Refresh your understanding of [[DNA Melting Thermodynamics]].
 +
# Complete the Simulating DNA Melting homework using [[DNA Melting: Simulating DNA Melting - Basics]] and learn more about the signals you will observe in the lab and how to start to analyze them.
 +
# Follow the guidelines in [[DNA Melting Part 1: Measuring Temperature and Fluorescence|Part 1]] of the lab 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 [[DNA Melting Report Requirements for Part 1|Part 1 of your lab report]].
 +
# Improve your simulation of DNA Melting results by adding the modeled effects described in [[DNA Melting: Model function and parameter estimation by nonlinear regression]].
 +
# Improve your instrument by adding temperature control and lock-in signal processing, as outlined in [[DNA Melting Part 2: Lock-in Amplifier and Temperature Control|Part 2]] of the lab.
 +
# Verify the performance of your instrument with known samples.
 +
# Measure your unknown samples.
 +
# Attend the tutorial on multi-parameter, nonlinear regression to further improve your model toward even better estimates of ''&Delta;H&deg;'', and ''&Delta;S&deg;'', and use to analyze your data from Part 2. Your goal is to identify the unknown sample.
 +
# Turn in [[DNA Melting Report Requirements for Part 2|Part 2 of your lab report]]. In this final report submission include Part 2 only, but note any significant revisions to hardware, software or methods that you may have made since Part 1.
  
 
==Objectives and learning goals==
 
==Objectives and learning goals==
Line 68: Line 86:
 
*[[DNA Melting: Simulating DNA Melting - Basics]]
 
*[[DNA Melting: Simulating DNA Melting - Basics]]
 
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]
 
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]
−
*[[DNA Melting Report Requirements for Part 1]]
 
 
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]
 
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]
−
*[[DNA Melting Report Requirements for Part 2]]
 
  
 
==Suggested readings and references==
 
==Suggested readings and references==

Latest revision as of 03:39, 13 April 2016

20.309: Biological Instrumentation and Measurement

ImageBar 774.jpg


DNA Excitation.JPG Example Melting Curve.jpg
Temperature cycling fluorometer apparatus. Example DNA melting curves showing the effect of varying ionic strength.

Overview

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 and Oligocalc.

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.[1]

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.[2] 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.[3]

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.

Instrument

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.
  • 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

DNA Melting Lab Part 1.png

  1. Refresh your understanding of DNA Melting Thermodynamics.
  2. Complete the Simulating DNA Melting homework using DNA Melting: Simulating DNA Melting - Basics and learn more about the signals you will observe in the lab and how to start to analyze them.
  3. Follow the guidelines in Part 1 of the lab to build a system for exciting, heating, measuring fluorescence, and measuring temperature of a DNA sample.
  4. Troubleshoot and optimize your instrument.
  5. Measure the signal to noise ratio.
  6. Generate melting curves for a known sample.
  7. Estimate the melting temperature of the known sample. Turn in Part 1 of your lab report.
  8. Improve your simulation of DNA Melting results by adding the modeled effects described in DNA Melting: Model function and parameter estimation by nonlinear regression.
  9. Improve your instrument by adding temperature control and lock-in signal processing, as outlined in Part 2 of the lab.
  10. Verify the performance of your instrument with known samples.
  11. Measure your unknown samples.
  12. Attend the tutorial on multi-parameter, nonlinear regression to further improve your model toward even better estimates of ΔH°, and ΔS°, and use to analyze your data from Part 2. Your goal is to identify the unknown sample.
  13. Turn in Part 2 of your lab report. In this final report submission include Part 2 only, but note any significant revisions to hardware, software or methods that you may have made since Part 1.

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.

Lab manual sections

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.


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