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

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*[[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 Report Requirements for Part 1]]
*[[DNA Melting: Simulating DNA Melting - Intermediate Topics]]
 
 
*[[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]]
 
*[[DNA Melting Report Requirements for Part 2]]

Revision as of 20:44, 22 July 2013

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.

Complementary 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 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, and measure the signal to noise ratio.
  5. Generate melting curves for a known sample.
  6. Estimate the melting temperature of the known sample. Turn in Part 1 of your lab report.
  7. Improve your understanding of noise sources and other non-idealities by completing additional simulation in DNA Melting: Simulating DNA Melting - Intermediate Topics.
  8. Improve your instrument by adding lock-in signal processing and temperature control, as outlined in Part 2 of the lab.
  9. Verify the performance of your instrument with the known sample.
  10. Measure your known and unknown samples.
  11. Attend the tutorial on multi-parameter, nonlinear regression to estimate ΔH°, and ΔS°, and use to analyze your data from Part 2.
  12. Turn in 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.

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.

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.