Dissociation%20Polynucleotides%20Lab%20Protocol%202009%20Spring

Dissociation%20Polynucleotides%20Lab%20Protocol%202009%20Spring

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1 BME 100L. Modeling Cellular and Molecular Systems Dissociation of Double Stranded Polynucleotides Objective: Inosinic acid (I) is a derivative of adenosine and pairs with cytidine (C), adenosine (A), and uridine (U). In this lab, we will determine the dissociation of double-stranded chains of polyinosinic acid-polycytidinic acid (polyI-polyC) as a function of temperature. “Dissociation” means breaking hydrogen bonds between complementary double-stranded nucleotide chains, resulting in two single-stranded chains. The structure of polyI-polyC is shown below. Note the locations of hydrogen bonds. O O P OH O N N N NH Inosinic acid Cytidinic acid O O P H N 2 Hydrogen bonds Dissociation (double-stranded to single-stranded conformation) can be measured by taking advantage of the hypochromicity of the polynucleotides (i.e. increased light absorption as double-stranded polyI-polyC chains become single-stranded during heating). Background: Polynucleotide strands are held together by covalent phosphodiester bonds between nucleotides (note the phosphate groups shown on each representative single strand in the figure above). This is often called the “sugar-phosphate backbone” of each single strand of polynucleotides in a double-stranded molecule. DNA primary structure is formed by phosphodiester bonds between the following nucleotides: Guanine (G), Adenine (A), Cytosine (C), and Thymine (T).
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2 Similarly, RNA primary structure is formed by phosphodiester bonds between nucleotides. However, Uracil is used instead of Thymine in RNA. (Figure from http://ghr.nlm.nih.gov) The most thermodynamically stable conformation of polynucleotides is in double- stranded form, where complementary bases on separate strands are paired by hydrogen bonds. “Complementary” refers to the nitrogenous bases that form hydrogen bonds with each other. In DNA, A and T are complementary (form two hydrogen bonds between them), and G and C are complementary (form three hydrogen bonds between them). This is often written as A::T and G:::C. The hydrogen bonding is called “base pairing.” This results in formation of the famous double helix (see figure below). Thymine Guanine Adenine Cytosine Uracil (Figures from the Nucleic Acid Database http://ndbserver.rutgers.edu)
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3 Although RNA is a single-stranded molecule, it is more thermodynamically stable when intrastrand base pairs form. RNA molecules form double-stranded regions by base pairing (A::U and G:::C), forming secondary structures within a single strand of RNA.
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This note was uploaded on 08/03/2009 for the course BME 100 taught by Professor Yuan during the Spring '07 term at Duke.

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Dissociation%20Polynucleotides%20Lab%20Protocol%202009%20Spring

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