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

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Complimentary DNA oligos in solution exist in a temperature dependent equilibrium between double stranded helicies and single stranded random coils. In this lab, you will build an instrument to measure the fraction of dsDNA in a sample as a function of temperature. The resulting plot of dsDNA fraction versus temperature is called a melting curve. Thermodynamic properties of the DNA annealing reaction, including ''ΔH°'', and ''ΔS°'', and the melting temperature can be extracted from the melting curve. The thermodynamic parameters depend on the sequence length, salt ion concentration, and degree of complementarity between the two oligos. 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.   
 
Complimentary DNA oligos in solution exist in a temperature dependent equilibrium between double stranded helicies and single stranded random coils. In this lab, you will build an instrument to measure the fraction of dsDNA in a sample as a function of temperature. The resulting plot of dsDNA fraction versus temperature is called a melting curve. Thermodynamic properties of the DNA annealing reaction, including ''ΔH°'', and ''ΔS°'', and the melting temperature can be extracted from the melting curve. The thermodynamic parameters depend on the sequence length, salt ion concentration, and degree of complementarity between the two oligos. 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) <ref name="SYBR Green Paper">[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=484200 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''':e103–10.]</ref>. 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 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.  
  
 
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 theoretical models and you will attempt to identify unknown samples.
 
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 theoretical models and you will attempt to identify unknown samples.

Revision as of 05:35, 29 August 2011

20.309: Biological Instrumentation and Measurement

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DNA Melting Apparatus

Introduction

Example DNA melting curves showing the effect of varying ionic strength. The data has been filtered to reduce noise.
Differentiating the melting curve simplifies finding Tm.

Complimentary DNA oligos in solution exist in a temperature dependent equilibrium between double stranded helicies and single stranded random coils. In this lab, you will build an instrument to measure the fraction of dsDNA in a sample as a function of temperature. The resulting plot of dsDNA fraction versus temperature is called a melting curve. Thermodynamic properties of the DNA annealing reaction, including ΔH°, and ΔS°, and the melting temperature can be extracted from the melting curve. The thermodynamic parameters depend on the sequence length, salt ion concentration, and degree of complementarity between the two oligos. 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 emitted by an excited sample.

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 theoretical models and you will attempt to identify unknown samples.

For more information about this lab, such as the fundamentals of DNA melting and fluorescent reporters involved, plus applications such as PCR, review the links to websites, videos, and academic papers found here: Resource list:DNA melting and PCR

How to do this lab

  1. Refresh your understanding of DNA Melting Thermodynamics
  2. Follow the guidelines in Part 1 of the lab manual to build a system for exciting, heating, measuring fluorescence, and measuring temperature of a DNA sample.
  3. Troubleshoot and optimize your instrument.
  4. Generate a melting curve for a known sample.
  5. Estimate the melting temperature of the known sample. Turn in Part 1 of your lab report.
  6. Improve your instrument by adding lock-in signal processing and temperature control, as outlined in Part 2 of the lab manual
  7. Verify the performance of your instrument with a known sample.
  8. Measure unknown samples.
  9. Use nonlinear regression to estimate ΔH°, and ΔS°, as outlined in the data analysis guidelines
  10. Turn in Part 2 of your lab report

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:e103–10.[1]

A more complete DNA melting and PCR resource list is available on this wiki. Please improve the page by adding relevant, high-quality sources.

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.

References

  1. 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:e103–10.