Lab: DNAIn this lab, you will learn about the structure of the DNA molecule, how DNA replicates itself, and some of the history behind the discovery of the structure of DNA.Introduction:Swiss chemist, Friedrich Miescher, isolated DNA from fish sperm and the pus of open wounds in 1869. Since it came from nuclei, Miescher named this new chemical, nuclein. Subsequently, the name was changed to nucleic acid and lastly to deoxyribonucleic acid (DNA). Chemist Robert Feulgen, in 1914, discovered that fuchsin dye stained DNA. DNA was then found in the nucleus of all eukaryotic cells.During the 1920s, biochemist P.A. Levene analyzed the components of the DNA molecule. He found it contained four nitrogenous bases: cytosine, thymine, adenine, and guanine; deoxyribose sugar; and a phosphate group. He concluded that the basic unit (nucleotide) was composed of a base attached to a sugar and that the phosphate also attached to the sugar. The nucleotide is the fundamental unit (monomer) of the nucleic acid polymer. There are four nucleotides: those with cytosine (abbreviated with the letter C), those with guanine (G), those with adenine (A), and those with thymine (T). Structure of a nucleotideDuring the 1920s, Frederick Griffith studied the differences between a disease-causing strain of the pneumonia, causing bacteria (Streptococcus pneumoniae), and a strain that did not cause pneumonia. Griffith, in 1928, was able to induce a nonpathogenic strain to become pathogenic with what he called a transforming factor.In 1944, Avery, MacLeod, and McCarty revisited Griffith’s experiment and concluded the transforming factor was more like DNA than like protein. Their evidence was strong but not totally conclusive. The then-current favorite for the hereditary material was protein; DNA was not considered by many scientists to be a strong candidate.The breakthrough in the quest to determine the hereditary material came from the work of Delbruck and Luria in the 1940s. Bacteriophages are a type of virus that attacks bacteria; the viruses that Delbruck and Luria worked with were those attacking Escherichia coli, a bacterium found in human intestines. Bacteriophages consist of protein coats covering DNA. Bacteriophages infect a cell by injecting DNA into the host cell. This viral DNA then ‘disappears’ while taking over the bacterial machinery and begins to make new virus instead of new bacteria. After 25 minutes, the host cell bursts, releasing hundreds of new bacteriophages. Phages have DNA and protein, making them ideal to resolve the nature of the hereditary material.In 1952, Hershey and Chase conducted a series of experiments to determine whether the protein or DNA was the hereditary material. By labeling the DNA and protein with radioisotopes, they would be able to determine which chemical (DNA or protein) was getting into the bacteria. Such material must be the hereditary material (Griffith’s transforming agent). Since DNA contains Phosphorous (P) but no Sulfur (S), they tagged the DNA with radioactive Phosphorous-32. Conversely, protein lacks P but does have S, thus it could be tagged with radioactive Sulfur-35.Hershey and Chase found that the radioactive S remained outside the cell while the radioactive P was found inside the cell, indicating that DNA was the physical carrier of heredity.Erwin Chargaff analyzed the nitrogenous bases in many different forms of life, concluding that the amount of purines does not always equal the amount of pyrimidines.DNA had been proven as the genetic material by the Hershey-Chase experiments, but how DNA served as genes was not yet certain. DNA must carry information from parent cell to daughter cell. It must contain information for replicating itself. It must be chemically stable, relatively unchanging. However, it must be capable of mutational change. Without mutations there would be no process of evolution.Many scientists were interested in deciphering the structure of DNA; among them were Francis Crick, James Watson, Rosalind Franklin, and Maurice Wilkens. Watson and Crick gathered all available data in an attempt to develop a model of DNA structure. Franklin took X-ray diffraction photomicrographs of crystalline DNA extract, the key to the puzzle.The data known at the time was that DNA was a long molecule, proteins were helically-coiled (as determined by the work of Linus Pauling), Chargaff’s base data, and the x-ray diffraction data of Franklin and Wilkens.Rosalind Franklin had significant contribution to determining the structure of DNA. However, some say that she does not get the credit that she deserves, while others argue that it is part of collaboration. You might check out these sites to learn more.National Institute of Health: Rosalind Franklin Chemical Heritage Foundation: DNADNA is a double helix, with bases to the center (like rungs on a ladder) and sugar-phosphate units along the sides of the helix (like the sides of a twisted ladder). The strands are complementary (deduced by Watson and Crick from Chargaff’s data: A pairs with T and C pairs with G, the pairs held together by hydrogen bonds). Notice that a double-ringed purine is always bonded to a single ring pyrimidine. Purines are Adenine (A) and Guanine (G). We have encountered Adenosine triphosphate (ATP) before, although in that case the sugar was ribose, whereas in DNA it is deoxyribose. Pyrimidines are Cytosine (C) and Thymine (T). The bases are complementary; with A on one side of the molecule you only get T on the other side. If we know the sequence of one strand, we know its complement.DNA was proven as the hereditary material by the work of Hershey and Chase. Watson and Crick had deciphered the structure of DNA. What remained was to determine how DNA copied its information and how that was expressed in the phenotype.Meselson and Stahl designed an experiment to determine how DNA replicated. The Meselson-Stahl experiment involved the growth of E. coli bacteria on a growth medium containing heavy nitrogen (Nitrogen-15 as opposed to the more common but lighter isotope, Nitrogen-14). The first generation of bacteria was grown on a medium where the sole source of N was Nitrogen-15. The bacteria were then transferred to a medium with light (Nitrogen-14) medium. Watson and Crick predicted that DNA replication was semi-conservative. If it was, then the DNA produced by bacteria grown on light medium would be intermediate between heavy and light. It was. DNA replication, shown in Figures 5 and 6, involves many building blocks, enzymes and a great deal of ATP energy.However, this process only occurs in a cell once per cell generation. Nucleotides have to be assembled and available in the nucleus, along with energy to make bonds between nucleotides. DNA polymerases unzip the helix by breaking the H-bonds between bases. Once the polymerases have opened the molecule, an area known as the replication bubble forms (always initiated at a certain set of nucleotides, the origin of replication). New nucleotides are placed in the fork and link to the corresponding parental nucleotide already there (A with T, C with G).Prokaryotes open a single replication bubble, while eukaryotes have multiple bubbles. The entire length of the DNA molecule is replicated as the bubbles meet.Objectives:Learn about the structure of the DNA molecule and how DNA replicates itselfThe nature of complementary DNA basesThe personalities and cultural context of the race to discover the structure of DNATime Requirements:This lab should take two hours to complete.Procedure:Download the DNA Lab Report and answer the questions.Expansion: Use these resources to develop an e-poster of a scientist of your choice who made contributions to our knowledge of the molecular basis of inheritance.
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