X-Ray Diffraction and the Structure of DNA

Advanced Preparation

The teacher needs to purchase the gum drops (20 per pair of students) and toothpicks. Time needed to divide up the materials is 15 minutes/ class.

Introduction

The English physicist Sir William Henry Bragg, b. July 2, 1862, d. Mar. 12, 1942, pioneered the determination of crystal structure by X-RAY DIFFRACTION methods, for which he and his son William Lawrence BRAGG received the 1915 Nobel Prize for physics. After Wilhelm Roentgen discovered X rays in 1895, Bragg began a lifelong investigation of the nature of radiation, principally X rays but also alpha and beta particles and gamma rays. After the discovery of the diffraction of X rays by crystals in 1912, Bragg and his son, William L., derived Bragg's law, which relates the wavelength of X rays to the glancing angle of reflection. In 1913 the elder Bragg built the first X-ray spectrometer, which he initially used to study X-ray spectral distributions. Within several years the they were able to use this instrument and Bragg's law to derive the structure of crystals and show the exact positions of atoms. Subsequently, they demonstrated that the properties and behavior of a large variety of substances can be related to the position of their constituent atoms.

William Lawrence Bragg went on to become director of the Cavendish Laboratory in Cambridge England. It was at this lab, while he as director, in the early 1950's that J.D. Watson and F.H. C. Crick, using the X-ray diffraction techniques that Bragg pioneered, deduced the double helical structure of Deoxyribonucleic acid. (DNA)

Biotechnology has many forms today: Crime detection, forensic science, recombinant DNA to strengthen plants and animals as well as fetal analysis to detect birth defects just to name a few. None of these would be possible if Watson and Crick had not deduced the structure of DNA using X-ray diffraction. Just as the engineer who is building a tall building must understand steel and cement, a scientist must understand the double helix before she can build a molecule.

In the last lab, we learned that for diffraction to work, the slits must be approximately the same width as the wavelength of the waves passing through it. In a real molecule the atoms are very close to each other. In a typical molecule the atoms are about 30 Angstroms apart. An angstrom is 10-10m. Using the figure on the next page as a reference, answer the following questions.

1. Would visible light be a good choice to diffract in a molecule? Why or why not?

No the wavelength is too long and the light waves would not penetrate the molecule.

2. If you had to choose one of the following to diffract in a molecule:

Radio waves, Microwaves, Infrared, Visible, Ultraviolet or X-ray, which would you choose and why?

In order to penetrate the molecule the wavelength of the light

needs to be approximately the same as the distance between the molecules.

X-rays have the appropriate wavelength to move through the molecule.

3. How would you detect the wave you chose in answer 2? Could you use your eyes?

You could use X-ray film, or a Geiger counter to detect the waves. They are not visible to the eye, the wavelength is too short.

Fig. 1. Electromagnetic Spectrum

Bragg's law allows us to determine the structure of three dimensional molecules using X ray diffraction. Bragg's law is illustrated below.

Bragg's law. The top surface represents one layer of atoms. The heavy line underneath represents a second layer of atoms. The path of the wave reflected off of the top surface is shorter than the path of the wave reflected off of the second surface. The second wave has to travel 2dSIN[[theta]] farther than the first wave did.

4. Color the part of the lines that represent the extra length the second wave has to travel

Bragg's law is:

      2dSIN[[theta]]=       
       m[[lambda]]

where d is the distance between atoms, [[theta]] is the angle of incidence and reflection with respect to the normal, [[lambda]] is the wavelength of the X-ray or light and m is a whole number integer (1, 2, 3,...)

Student Objectives

* Students will interpret a simulated X-ray crystallography diagram to determine the

structure of DNA .

Materials

1. toothpicks, 10 per student group

2. gumdrops, 20 per student group

3. bamboo skewers, 10 per student group

Procedure:

The picture below is similar to the one that Watson and Crick observed to confirm the double helical structure of DNA. Answer the following questions by looking at the picture.

Figure 1. X-ray diffraction of DNA molecule

1. Does this pattern suggest a regular repeating pattern or an irregular, non-repeating pattern. Explain.

The pattern appears to be a regular pattern with an outward spiral

2. Assume the wavelength of the X-rays used were 1 X 10-10 m, and the angle of incidence is 8.45o to the first line of reinforcement (m=1). How far is it in between bases on a DNA molecule?

The distance is about 3.4 Angstroms ( 3.4 x 10-10 m, or 340 nanometers)

3. Using gum drops and tooth picks, and bamboo skewers, build a model of DNA with 10 bases on each strand. Thus you will need 20 gum drops, 20 toothpicks and 10 bamboo skewers.

* Pick a color of gum drop to represent A, T, G and C. Record in the table.

* Make one strand of DNA have the following sequence: A,T,G,C,C,G,T,A,A,T

* REMEMBERING that A pairs with T, and G with C, make the complementary strand of DNA.

* Adjust the distance between gum drops on the same strand to 3.4 cm.

* Using bamboo skewers, connect the two strands 12 cm apart. Your molecule should look like the picture below.

Base                                    Color                                   
Adenine                                                                         
Guanine                                                                         
Cytosine                                                                        
Thymine

4. Now hold your DNA molecule from the top and bottom, and twist it so that it makes 1 complete turn (180o). See picture below.

5. Observe your DNA looking down the central axis. How many base pairs do you see before they begin to overlap?

You can see approximately 5 sets of base pairs.

6. Draw the atoms from this view and relate it to Figure 1 (X-ray diffraction pattern of DNA)

Drawings should resemble two rows of atoms, spiraling away at a set distance from one another, the double helix.

Conclusion

Explain how the distance between the two slits can be determined without actually seeing the slits, and how the structure of DNA can be determined without actually seeing the atoms themselves. What model was used by Bragg to develop his law of diffraction, and how did this lead to the discovery of the structure of DNA.

Using Bragg's law the student scan determine the distance d between slits (or

atoms) from the formula

2dsin[[theta]]= m[[lambda]], d = m[[lambda]] where [[theta]] [[iota]]s the angle of incidence

2sin[[theta]] [[lambda]] is the wavelength of the light

m is a whole number integer.

The X-ray crystallography process creates a scan or 'picture' of the DNA molecule. which shows the placement of the base pairs. It is possible to visualize the placement of the amino acids and then he structure of the DNA molecules

Braqg used the double slit diffraction model to develop his equation for the distances between molecules. This allowed him to substitute the distance between atoms,

or in this case the base pairs, for the distance in the double slit equation. He found the value for d from the equation 2dsin[[theta]]= m[[lambda]], d = m[[lambda]] 2sin[[theta]]

Knowing the distance between the base pairs, made it possible determine the placement of the amino acids and build a model of a DNA molecule.