To gauge the fidelity of DNA synthesis, Arthur Kornberg and co...

Question

To gauge the fidelity of DNA synthesis, Arthur Kornberg and colleagues devised a technique called nearest-neighbor analysis, which determines the frequency with which any two bases occur adjacent to each other along the polynucleotide chain (J. Biol. Chem. 236: 864–875). This test relies on the enzyme spleen phosphodiesterase (see the previous problem). DNA is synthesized by polymerization of 5'-nucleotides—that is, each nucleotide is added with the phosphate on the deoxyribose. However, as shown in the accompanying figure, the phosphodiesterase enzyme cleaves DNA between the phosphate and the C-5' atom, thereby producing 3'-nucleotides. In this test, the phosphates on only one of the four nucleotide precursors of DNA (cytidylic acid, for example) are made radioactive with ³²P, and DNA is synthesized. Then the DNA is subjected to enzymatic cleavage, in which the radioactive phosphate is transferred to the base that is the 'nearest neighbor' on the 5' side of all cytidylic acid nucleotides.

Diagram showing DNA synthesis from 32P-labeled nucleotides and enzyme cleavage transferring radioactive phosphate to adjacent bases.

Following four separate experiments, in each of which a different one of the four nucleotide types is radioactive, the frequency of all 16 possible nearest neighbors can be calculated. When Kornberg applied the nearest-neighbor frequency test to the DNA template and resultant product from a variety of experiments, he found general agreement between the nearest-neighbor frequencies of the two. Analysis of nearest-neighbor data led Kornberg to conclude that the two strands of the double helix are in opposite polarity to one another. Demonstrate this approach by determining the outcome of such an analysis if the strands of DNA shown here are (a) antiparallel versus (b) parallel:

Diagram comparing DNA strands in (a) antiparallel and (b) parallel orientations with nucleotide sequences and phosphate links.

Accepted Answer

Everyone Welcome back, let's look at our next question. It says corn berg was able to compare the nitrogenous based compositions of the DNA template with the DNA product. What was the result of his analysis? We can recall the corn berg, Did much of the research showing how cells actually make D. N. A. In the early days of discovery about the nature of D. N. A. And he compared when you have the template and when you have the product, how much of each base did you have making up these two things and compare them? And the result of his analysis showed choice. See the synthesized DNA products were a faithful copy of the DNA template. He showed within experimental error that there were equal amounts of A. T. C. And G. In the template and the product. So that eliminated the possibility um that we see here that the products were longer because you didn't see more of these bases in the product or that they were shorter since you didn't see less of them or that they were in random lengths. You saw that they were equal to the template. So that began this process of showing that D. N. A. Was made in faithful copies of the template. See you in the next video

Key Concepts

DNA Structure and Antiparallel Strands

Nearest-Neighbor Analysis

Phosphodiester Bond and Enzymatic Cleavage

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