Reyes, Sashley Paringit, Jasmine Bijay Goseingfiao, Jenifer Leano, Florante Albano, Kevin
EXPERIMENT X NUCLEIC ACIDS OBJECTIVES: 1) To isolate RNA from yeast. 2) To test the properties of the isolated RNA. MATERIALS: Reagents
Instruments
METHODS: I.
Isolation of RNA from Yeast
II.
Qualitative Tests for Nucleic Acids A. Test for Nucleoproteins B. Milc Acid Hydrolysiss a.
Test for Inorganic Phosphates
b. Test for the Presence of Pentoses c.
Test for the Presence of Purines
RESULT AND DISCUSSION: Nucleic acids are made up of nucleotides. A component of each nucleotide is the purine base (adenine or guanine) or pyrimidine base (cytosine, uracil or thymine), pentacarbon sugar: ribose or deoxyribose and orthophosphoric acid residue. The base binds the sugar by the β-N-glycosidic β-N-glycosidic bond, orthophosphoric acid residue binds the sugar component by an ester bond through the -OH group at carbon 3' or 5' of ribose or deoxyribose. Individual nucleotides are bound with phosphodiester bonds between carbons 3' and 5’.
The acidic character of the nucleic acids is caused by orthophosphate residues, each of which contains H+ capable of dissociation. Due to this, nucleic acids are polyanions - carriers of many negative charges, and this makes them capable of interacting with polycations, particularly with alkaline proteins - which are carriers of positive charges. They also bind with micromolecular compounds of an alkaline nature, such as methylene blue. In the natural environment, DNA mainly binds with alkaline proteins - histones, whereas RNA mainly binds with neutral proteins, which are part of the ribosome. Complexes of nucleic acids with proteins are called nucleoproteins. In the student laboratory conditions, artificial nucleoproteins can be obtained by mixing a solution of nucleic acid with blood serum. Nucleoproteins dissociate into its constituents in concentrated salt solutions. In an alkaline environment, nucleic acids form salts - nucleates, which are soluble in water. They can be precipitated from solution wit h ethanol. Sugar constituents of nucleic acids: ribose and deoxyribose can be detected directly in the solutions of these acids or their salts without w ithout prior hydrolysis. Ribose contained in the RNA, purine nucleosides and nucleotides, heated with concentrated HCl dehydrates to furfural, which with orcin forms a complex of a stable green colour. Deoxyribose, contained in the D NA, when heated with concentrated sulphuric acid is converted into hydroxylevulinyl aldehyde. This compound forms a blue colour complex in reaction with diphenylamine. Purine bases can only be detected in the hydrolysis products of nucleic acids. The sample of nucleic acid, intended for the detection of purines, should be hydrolysed in sulphuric acid at 100°C. Nucleic acids are hydrolysed, initially to mononucleotides. Purine mononucleotides are further hydrolysed to bases, pentoses and orthophosphoric acid. The action of this acid leads to the hydrolytic breakdown of the β-N-glycosidic β-N-glycosidic bonds between the purine and the ribose or deoxyribose. The following purine bases are released: adenine and guanine. Purine bases precipitate e asily as insoluble complexes with the ions of copper or silver. In the same conditions, pyrimidine nucleotides are stable and do not decompose.
Name of Test Biuret Test for Nucleoproteins
Observation ++ Purple with blue precipitates
Test for Phosphates (Acid Hydrolysate)
+ Yellow solution with yellow precipitates
Test for Sugar 0.1% Ribose
+++ Olive green solution
0.1% Glucose Acid hydrolysate
++ Yellow clear solution ++ Olive green solution
Test for Purine Bases
+ White precipitates in foamy
Explanation Formation of purple colored product due to the reduction of Cu2+ to Cu+ Hydrolysis of pyrophosphate to phosphate forming yellow precipitate Pentose is dehydrated to furfural and reacts with orcinol to form green product No reaction with orcinol Pentose is dehydrated to furfural and reacts with orcinol to form green product Hydrolysis of N-beta-glucosidic
solution
bonds between purine and ribose result in a release of of purine bases (A and G) caused by NH4OH Ag+ precipitate cause the formation of foamy gelatinous substance
APPLICATION Used in nucleic acid testing: -
Blood screening
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Detecting infectious disease
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Predicting cancer and guiding cancer treatment
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For personalized and precision medicine
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Screening for genetic disorders
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Diagnosis of mitochondrial diseases
QUESTIONS 1) What are the purine bases found in nucleic acids?
Adenine = 6-amino purine
Guanine = 2-amino-6-oxy purine
Hypoxanthine = 6-oxy purine
Xanthine = 2,6-dioxy purine
2) How do you account for the formation of precipitates in the test for purines?
