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

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*[[DNA Melting Report Requirements for Part 2]]
 
*[[DNA Melting Report Requirements for Part 2]]
 
*[[DNA Melting: Simulating DNA Melting - Intermediate Topics]]
 
*[[DNA Melting: Simulating DNA Melting - Intermediate Topics]]
*[[DNA Melting Report Requirements for Part 3]]
 
 
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]
 
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]
 
*[[DNA Melting Part 5: Multi-parameter Data Analysis]]
 
*[[DNA Melting Part 5: Multi-parameter Data Analysis]]
*[[DNA Melting Report Requirements for Part 5]]
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*[[DNA Melting Report Requirements for Part 2]]
  
 
==Suggested readings and references==
 
==Suggested readings and references==

Revision as of 17:52, 26 September 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.

How to do this lab

  1. Refresh your understanding of DNA Melting Thermodynamics
  2. Complete the Simulating DNA Melting homework in Part 1 of the lab and learn more about the signals you will observe in the lab.
  3. Follow the guidelines in Part 2 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. Generate a melting curve for a known sample that we provide.
  6. Estimate the melting temperature of the known sample. Turn in Part A of your lab report.
  7. Improve your understanding of noise sources and other non-idealities by completing additional simulation in Part3 of the lab.
  8. Formalize your understanding on paper and turn in Part B of your lab report.
  9. Improve your instrument by adding lock-in signal processing and temperature control, as outlined in Part 4 of the lab.
  10. Verify the performance of your instrument with the known sample.
  11. Measure your unknown samples.
  12. For Part 5, use multi-parameter, nonlinear regression to estimate ΔH°, and ΔS°, as outlined in the data analysis guidelines.
  13. Turn in Part C of your lab report. In this final report submission, include Parts A and B and note any significant revisions that you may have made.

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.

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.