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Stretching Lambda Digest DNA with the MFP-1D

The familiar double-stranded helix structure of DNA was first proposed by Watson and Crick in 1953.1  The Watson-Crick conformation (labeled "B-DNA") with its characteristic 10.5 base-pairs per helix turn is only one of numerous possible conformations. A number of factors can contribute to the conformation of DNA including chemical denaturants, pH, temperature and force. In 1996, Smith et al. stretched a strand of B-DNA that was attached to a glass slide and a magnetic bead. They discovered that applying a force of 65 pN resulted in a characteristic plateau in the force vs. distance curve consistent with a structural reordering.2 More recently, Rief et al. observed a transition where the double stranded (ds) DNA was melted into a single stranded (ss) DNA.3

In this work, the MFP-1D has been used to study the mechanical properties of double stranded (ds) l-digest DNA. The figure above shows three pulls made on a single DNA molecule. The graph shows a force measurement of a single tethered molecule of Lambda Digest DNA showing the B-S and the melting transition. During the extension of the molecule (red trace), the DNA first goes through the B-S transition (the plateau), and then melts to single-stranded DNA (ssDNA) at a higher force. During relaxation of the molecule (blue trace), the DNA doesn't reanneal so the curve is a simple freely-jointed-chain curve indicative of ssDNA.  The traces were made at a pulling speed of 1m/second. Data courtesy of H. Clausen-Schaumann and R. Krautbauer, Gaub Lab, LMU-München. The cantilever was a 310 mm long, triangular SiN cantilever with a spring constant of 12 pN/nm.4

To find out more about recent force measurements on DNA including sequence dependent forces, see reference 5 below. 

DNA-deposited Slides for Force Measurements and Other Biological Experiments
If you would like to perform the above experiment, Asylum Research now provides DNA bound to gold on glass slides, along with other gold slides for your experiments.The DNA slide is prepared with Lambda Digest DNA. This is a good sample for observing the 65 picoNewton B-S transition in single molecule pulling experiments.2 The B-S transition provides a useful standard for checking the performance and calibration of single molecule force measurements. In addition to the B-S transition, the melting transition can be observed at a force of roughly 150 picoNewtons.5

1. J. D. Watson and F. H. C. Crick, The Structure of DNA.  Cold Spring Harbor Symp. Quant. Biol.18, 123 (1953). and J. D. Watson and F. H. C. Crick, Molecular Structure of Nucleic Acids - The Structure of DNA. Nature 171, 737 (1953).
2.S.B. Smith, Y. Cui and C. Bustamante, Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules. Science 271, 759 (1996).
3. H. Clausen-Schaumann, M. Rief, C. Tolsdorf and H. E. Gaub, Biophysical Journal 78, 1997 (2000).
4. Park Scientific Microlevers
5. M. Rief, H. Clausen-Schaumann and H. E. Gaub, Nature Structural Biology 6, 346 (1999).

 

 

B-S melting transition of DNA.

 


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