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Determination of the Total DNA and RNA Content in Rat Liver, Kidney and Brain pre

Determination of the Total DNA and RNA Content in Rat Liver, Kidney and Brain pre

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Published by Karina Khan

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Published by: Karina Khan on Apr 02, 2011
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11/09/2012

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1
Name
: K***** K***
ID
:
Lab Partner
:
Date
: Wednesday 16
th
February 2011
Course Code:
BIOL2362  Further Metabolism and Gene Expression
Title
: Isolation, Quantification and Purity Determination of DNA from Rat Liver and Kidney andWhole Cows BloodDetermination of the Total DNA and RNA Content in Rat Liver, Kidney and Brain
Aim
: To extract DNA from cows blood, rat liver and rat kidney; to determine the quantity of DNAfrom Rat liver, kidney and brain using the diphenylamine reaction; to determine the quantity of RNA from rat liver, kidney and brain using the orcinol reaction; to quantify DNA using UVspectrometry and to determine the purity of various DNA samples.
Theory
:DNA is the chemical basis of heredity and is organized into genes; it directs the synthesis of RNA,which in turn directs protein synthesis (Murray, et al. 2003)A deoxyribonucleic acid (DNA) molecule consists of two long polynucleotide chains composed of four types of nucleotide subunits (Alberts, et al. 2006).The structure of a single DNA polynucleotide chain is shown in Figure 1.
 
Figure 1: Structure of a single polynucleotide strand of DNA(Nelson and Cox 2008).Phosphodiester bonds (one of which is shaded) link successivenucleotide units (Nelson and Cox 2008). Phosphodiester bonds join the 5'-hydroxyl group of thedeoxypentose of one nucleotide to the 3'-hydroxyl group of thedeoxypentose of an adjacent nucleotide through a phosphategroup (Harvey, Ferrier and Champe 2004).The nitrogenous bases present in DNA are derivatives of twoparent compounds, pyrimidine and purine (Nelson and Cox2008).
 
The bases present in DNA are adenine, thymine, guanineand cytosine. Hydrogen bonds between the base portions of the nucleotideshold the two chains together (Alberts, et al. 2006).The bases of one strand of DNA are paired with the bases of thesecond strand, so that an adenine is always paired with athymine and a cytosine is always paired with a guanine (Harvey,Ferrier and Champe 2004).
 
2Such base pairing results in one DNA strand being complementary to the other. Hydrogen bondingbetween the DNA bases can be seen in Figure 2.DNA consists of two associated polynucleotide strands that wind together to form a double helix.The two sugar phosphate backbones are on the outside of the double helix, and the bases project into the interior. The orientation of the two strands is anti-parallel; that is, their 5´
3´directionsare opposite. (Lodish, et al. 2007).
 
The structure of the DNA double helix is shown in Figure 2.DNA is present not only in chromosomes in the nucleus of eukaryotic organisms, but also inmitochondria and the chloroplasts of plants. Prokaryotic cells, which lack nuclei, have a singlechromosome, but may also contain DNA in the form of plasmids (Harvey, Ferrier and Champe2004).RNA is found in both the nucleus and the cytoplasm, and an increase in protein synthesis isaccompanied by an increase in the amount of cytoplasmic RNA and an increase in its rate of turnover (Nelson and Cox 2008).In all prokaryotic and eukaryotic organisms, three main classes of RNA molecules exist: messengerRNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA) (Murray, et al. 2003).The templates for protein synthesis are RNA (ribonucleic acid) molecules. In particular, a class of RNA molecules called messenger RNA (mRNA) are the information-carrying intermediates inprotein synthesis. Other RNA molecules, such as transfer RNA (tRNA) and ribosomal RNA (rRNA),are part of the protein-synthesizing machinery (Stryer, Berg and Tymoczko 2002).Figure 2: The Structure of the DNA double helix showinghydrogen bonding between the bases (Hames and Hooper2005).
 
3Figure 3: Structure of Ribose and Deoxyribose (Hamesand Hooper 2005).On the Carbon 2 position on the ribose sugar there is ahydroxyl group as compared to the hydrogen present onthe same position of the deoxyribose sugar.The primary structure of RNA is similar to that of DNA but with two exceptions: the sugarcomponent of RNA, ribose, has a hydroxyl group at the 2´ position (this can be seen in Figure 3) andthymine in DNA is replaced by uracil in RNA (Lodish, et al. 2007).Most RNA molecules are single-stranded but an RNA molecule may contain regions whichcan form complementary base pairing wherethe RNA strand loops back on itself (Hamesand Hooper 2005).This can be seen in Figure 4.Figure 4: Self-complementarity in RNA(Stryer, Berg and Tymoczko 2002) DNA can be chemically identified by the diphenylamine test. Diphenylamine reagent is preparedusing acetic acid and sulphuric acid. The structure of diphenylamine is shown in Figure 5.(Wood 2010)In contrast to the stability of the links between pyrimidines and deoxyribose, those betweenpurines and deoxyribose are very labile, so it appears that diphenylamine reacts with the sugarresidues originally combined with purines in the DNA (Burton 1955).Under extreme acidic conditions, DNA becomes depurinated (MoonHeart Infotech 2009)
 
Depurination is an alteration of DNA in which the purine base is removed from the deoxyribosesugar by hydrolysis of the beta-N-glycosidic link between them. After depurination, the sugarphosphate backbone remains and the sugar ring has a hydroxyl (-OH) group in the place of thepurine.Depurination of the DNA is followed by
he dehydration of sugar to hydroxylevulinylaldehydewhich in turn reacts with diphenylamine to give blue color (MoonHeart Infotech 2009).The intensity of the blue colour is proportional to the concentration of DNA in the sample and thiscould be measured via spectrophotometry.

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