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History of Molecular Genetics and Biotechnology Help (page 2)

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By — McGraw-Hill Professional
Updated on Apr 25, 2014

X-ray-diffraction

X-ray-diffraction data from crystalline materials has been critical for the elucidation of three-dimensional shapes of nucleic acids (e.g., DNA, tRNAs) and proteins (e.g., myoglobins, viral capsomeres, enzymes). This technique has been expanded to include the determination of multi-subunit molecules.

Chromatography

During the mid-1940s and early 1950s, various forms of chromatography were perfected, enabling molecules to be separated by differences in solubilities in organic solvents, electrical charge, molecular weight, and specific binding properties for the support medium, or combinations of these factors. Erwin Chargaff used paper chromatography to determine the base compositions of DNAs from various sources. He found that the molecular ratio of adenine was equivalent to that of thymine and the ratio of guanine equals that of cytosine. This was a vital clue for James Watson and Francis Crick in their search for the structure of DNA.

Bioinformatics

Automated equipment is now available for doing many repetitive biochemical tasks. DNA synthesizers can be programmed to make oligonucleotide (many nucleotides) sequences of any desired composition. Automated instrumentation is now available for isolating DNA, and sequencing DNA or protein fragments, as well as for performing enzyme assays and other tasks. Computer software has been developed to interpret data from electropherograms, and to search databases for similar or identical sequences (bioinformatics).

Techniques

Several techniques have been developed to separate, rejoin, synthesize, or break nucleic acid molecules. Separation of the complementary chains of a DNA molecule is known as denaturation. DNA is denatured if placed in alkali (0.2N NaOH) or when boiled. The latter process is referred to as melting. Separation of DNA strands can be detected by spectrophotometric instruments; optical density (OD) or absorbance at 260 nm increases during the melting process. The temperature at which the increase in OD260 is 50% of that attained when strand separation is complete is known as the melting temperature (Tm). Because G and C base-pair by three hydrogen bonds, whereas A and T base-pair by two hydrogen bonds, the higher the G-C content in DNA the higher the melting temperature. Melting is enhanced where there are clusters of A's and T's, and also when all the purines (A, G) are on one strand and all the pyrimidines (T, C) are on the other strand.

If DNA is boiled and then quickly cooled, the strands will remain single; if cooled slowly, complementary strands will base-pair and reform double-helical DNA molecules. This process is called renaturation or annealing. Hybrid DNA-RNA molecules can be produced by analogous processes from single strands. RNA can be totally hydrolyzed to nucleotides by exposure to high pH (alkali). This property can be used to purify DNA from amixture of DNA and RNA. Single-stranded DNA will bind to membranous filters made of nitrocellulose; RNA will pass through such filters. However, if single-stranded RNA is complementary to nitrocellulose-bound single strands of DNA, it will form DNA-RNA hybrid molecules and be retained by such a filter. This technique is used in various blotting procedures, such as Southern, Northern and Western blotting (see Section 7 later in this chapter).

There are two main methods for breaking long DNA molecules into fragments of suitable size for sequencing or for recombinant DNA techniques: (1) mechanical shearing and (2) restriction end onuclease treatment. If a solution of DNA is subjected to the stirring forces of a Waring blender or forced through a narrow tube, the ends of long DNA strands will usually move at different speeds; this stretches the DNA and tends to break it. This phenomenon is called shearing. The higher the stirring speed, or velocity, of flow through an orifice, the greater the shearing force. Shearing can also be achieved by exposure of a solution of DNA to ultrasound. The effectiveness of any shearing force increases with molecular size of the DNA, but decreases with concentration because entanglement of DNA molecules reduces the effective stretching. Restriction endonuclease treatment employs the use of enzymes that break the bonds between specific base-pair sequences of DNA. These enzymes will be discussed later in this chapter in the sections Nucleic Acid Enzymology, and Recombinant DNA Technology.

Radioactive Labels

Radioactive elements have historically been used as highly sensitive labels for detecting minute amounts of specific macromolecules. However, recent advances in fluorescent molecule technology (i.e., the development of fluorescent dyes to tag molecules of interest) have led to the replacement of many radioactive tracers by these fluorescent molecules. Fluorescent molecules are often safer to use, easier to dispose of and better for the environment.

EXAMPLE 12.1 A. D. Hershey and M. Chase differentially labeled the nucleic acid and the protein components of T2 phages. They used radioactive 32P in place of normal 32P to label DNA; radioactive 35S was used in place of normal 35S to label protein (cysteine and methionine are two amino acids that contain sulfur). Since there is no phosphorus in phage proteins and no sulfur in nucleic acids, the fate of both viral components could be followed during the viral life cycle. After allowing the phages to become attached to sensitive Escherichia coli host cells, the mixture was subjected to the shearing forces of a Waring blender. The mixture was centrifuged to sediment the cells and then activity characteristic of each radionuclide was assayed in the pellet and in the supernatant fluid. All of the 32P activity was found in the bacterial pellet and virtually all of the 35S was found in the supernatant. 32P was found in some progeny phages, but no 35S was found. The inference is that phages inject their DNA into host cells. Blender treatment shears the phage tail fibers from receptor sites on host cells; the empty phage protein capsids (ghosts) are therefore left free in the supernate. Semiconservative replication from the infecting 32P-labeled DNA caused some progeny phages to be released with one of the original radioactively labeled infecting strands. This experiment was the first to demonstrate that DNA, and not protein, is the genetic material in phages.

DNA labeled with radioactive elements can be revealed in a photographic technique called autoradiography. A preparation of DNA on a filter paper can be covered with a photographic film. As the radioactive elements undergo radioactive decay, they release charged particles that cause a chemical reaction on the film. After development of the X-ray film, the location of the labeled DNA is revealed by dark spots.

A radioactive isotope of phosphorus (32P) is also widely used to label nucleic acids; it emits a strong beta particle and has a half-life of 14.3 days. An instrument called a scintillation counter is used to detect radioactive disintegrations.

Any organic substance can be labeled with radioactive carbon (14C). This isotope emits a weak beta particle and has a relatively long half-life of 5730 years. All living organisms incorporate a predictable amount of 14C while alive. After death, 14C decays to 14N at the predictable rate of its half-life. This knowledge allows the dating of organic remains from the time of death up to about 40,000 years before the present.

Radioactive iodine (125I) has a half-life of about 60 days, emits γ-rays, and is used to label proteins. Highenergy γ-rays (gamma) can be detected by a crystal scintillation counter. A liquid scintillation counter must be used to detect weaker beta particles, although it can also detect γ-rays. This isotope is easily coupled to the amino acid tyrosine. Radioactive sulfur (35S) is used to label the amino acids cysteine and methionine; 35S can also be used to label nucleic acids. 35S is more desirable than 32P for most autoradiography because it has a half-life of 87.1 days and emits a much weaker beta particle that gives sharper bands. It is much less hazardous to handle than 32P and poses less waste-disposal problems.

Even nonradioactive isotopes (e.g., 15N) have been useful in solving fundamental problems in molecular biology.

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