Extraction and estimation of the nucleic acid-.pptx

The PPT described two well-established colorimetric methods for nucleic acid estimation are the Diphenylamine (DPA) method for DNA and the Orcinol method for RNA. These techniques utilize the unique chemical properties of sugar moieties in DNA and RNA to produce colorimetric reactions. The intensity of the resulting color, measurable by spectrophotometry, directly correlates with the nucleic acid concentration in the sample. As important analytical tools, these methods are crucial for accurately quantifying nucleic acids in various research and diagnostic applications, providing essential data for molecular biology studies. This presentation will provide a foundation for understanding the principles and applications of these methods in nucleic acid research. By mastering these techniques, students will gain valuable skills in nucleic acid analysis, essential for a wide range of molecular biology applications.

Nucleic acid estimation using DPA (Diphenylamine) and Orcinol methods involves colorimetric techniques to quantify DNA and RNA, respectively:

DPA Method for DNA: The DPA method is used to estimate DNA concentration. DPA reacts with deoxyribose sugar in DNA under acidic conditions to form a blue-colored complex. The intensity of this color, measured spectrophotometrically, correlates with the DNA concentration.

Orcinol Method for RNA: The Orcinol method is employed to estimate RNA concentration. Orcinol reacts with the ribose sugar in RNA, in the presence of ferric chloride and hydrochloric acid, producing a green-colored complex. The absorbance of this complex is measured spectrophotometrically to determine the RNA concentration.

Both methods are sensitive and specific to their respective nucleic acids.

1. Extraction and Estimation of the Nucleic Acid Karuna Irungbam Division of Biochemistry, ICAR-IVRI, Izatnagar , Bareilly

2. Introduction Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. The very first DNA isolation was done by a Swiss physician, Friedrich Miescher in 1869. Nucleic acid purification involves cell lysis to create a lysate, inactivating nucleases, and separating the desired nucleic acids from the lysate. Friedrich Miescher SOURCE: https ://en.wikipedia.org/wiki/ Friedrich_Miescher

3. Nucleic Acid Extraction

4.   Trichloroacetic acid Guanidinium Thiocyanate -Phenol-Chloroform Extraction Alkaline Extraction Method CTAB Extraction Method  Ethidium Bromide ( EtBr )- Cesium Chloride ( CsCl ) Gradient Centrifugation Purification of Poly (A) +  RNA by Oligp ( dT )-Cellulose Chromatography  Solid-phase Nucleic Acid Extraction Silica Matrices Glass Particle Diatomaceous Earth Magnetic Bead Based Nucleic Acid Purification Anion-Exchange Material Different Techniques Used:

5. Principle : Trichloroacetic acid (TCA) is commonly used to precipitate nucleic acids and to extract bases. Reagents : Trichloroacetic acid (TCA): 5% and 10% in distilled water Normal saline solution 0.9% NaCl in glass distilled water Extraction of Nucleic acid using TCA Method

6. Protocol Take 2 ml of homogenate in 15 ml of centrifuge tube 1 g of liver tissue is homogenized in 20 ml saline, filtered, and the supernatant is used for nucleic acid extraction. Add 5 ml of chilled 10% TCA and mix by pipetting up and down, Centrifuge at 1300g for 2 min Discard the supernatant, add 5 ml chilled 10% TCA to the pellet, and resuspend. Wash the pellet with 5 ml of 5% TCA, centrifuge at 1300g for 2 min, and transfer the supernatant to a fresh tube (extract 2). Centrifuge at 1300g for 2 min, discard the supernatant, wash the pellet with 10 ml of 95% ethanol, and centrifuge again at 1300g for 2 min. Repeat the ethanol wash. Discard the supernatant, add 5 ml of 5% TCA (RT), mix well, and incubate in a 90°C water bath for 15 min, agitating occasionally. Centrifuge at 1300g for 2 min, and transfer the supernatant to a fresh tube (extract 1).

7. Preparation of reagents Trituration of sample Filtration of homogenised sample Incubation with TCA Centrifugation and separation Preservation of supernatant

8. Estimation of DNA Using Diphenylamine Method

9. Principle : This reaction relies on the color change of deoxyribose in DNA with diphenylamine (DPA). The DPA reagent, containing acetic and sulfuric acids, cleaves DNA and hydrolyzes glycosidic bonds upon heating. The 2-deoxyribose is converted to ω- hydroxylevulinyl aldehyde, which reacts with diphenylamine to produce a blue color. The intensity of the blue solution indicates the DNA concentration. The reaction is non-specific to DNA and occurs with 2-deoxypentoses. In DNA, only deoxyribose of purine nucleotide reacts, representing half of the total deoxyribose. This consistency in both the standard and unknown samples allows direct concentration calculation from the standard graph. SOURCE : https ://d1vffmuvmgkypt.cloudfront.net/pdf/ridacom_ltd/himedia_laboratories/TD_HTBC006_pdf

10. Materials required: 1. Equipments: • Spectrophotometer • Water bath 2. Chemicals/reagents: •Standard DNA solution (1mg/ml) • Diphenylamine reagent • DNA sample in saline citrate buffer • Glacial acetic Acid • Concentrated H 2 SO 4 • Ethanal 3. Glasswares and others: • Test tubes • Pipettes • Graduated cylinder

11. Protocol (A). Preparation of reagents : 10% trichloroacetic acid (TCA) in distilled water. Diphenylamine reagent: Dissolve 1.5g diphenylamine in 100ml of glacial acetic acid. Add 1.5ml of conc H2SO4. Store the solution in a dark glass bottle. On the day of use, prepare a fresh solution of ethanal (1ml) in dH2O (50ml). Add 0.5ml of this solution to each 100ml of the diphenylamine solution. Note: Wear eye protection and use a fume cupboard when preparing this reagent. Diphenylamine is harmful if ingested or inhaled and may irritate skin or eyes if it comes into contact with them. 3. DNA standard: 20mg of DNA in 20ml of 10%TCA. Heat at 70°C for 15min and cool. Stock solution (1mg/ml) is than store at 4°C

12. (B). Assay : Prepare a series of standard DNA dilutions in water to achieve concentrations of 100-800 µg/ml as shown in the table. Add 2 ml of DPA to each dilution, blank, standard, and unknown sample, and mix well. Use Tube B as the blank and Tubes S1 through S5 for the standard calibration curve. Use Tubes Test 1-3 for unknown samples. Incubate all tubes in boiling water for 10 minutes. Cool the tubes and measure the absorbance at 595 nm against the blank. Construct a standard curve of absorbance (A595) vs. quantity of DNA. Calculate the concentration of the unknown DNA solution using the standard curve.

13. Tube Name Stock std ( ml of 1mg/ml) Distilled water (ml) Extract (ml) DPA reagent Mix vigorously and keep in boiling water bath for 10 min. Cool to room temp and measure absorbance at 595nm blank 1 2 S1 0.1 0.9 2 S2 0.2 0.8 2 S3 0.4 0.6 2 S4 0.6 0.4 2 S5 0.8 0.2 2 TEST1 0.5 0.5 2 TEST2 1.0 2 Protocol :

14. Preparation of reagents Preparation of stock solution Preparation of standards Incubation Transfer into cuvette Absorbance reading at 595nm Pictorial representation of the Protocol :

15. Tube Concentration ( ug /ml) Absorbance reading at 595nm blank 0.018 S1 100 0.149 S2 200 0.255 S3 400 0.45 S4 600 0.643 S5 800 0.804 TEST1 ? 0.137 TEST2 ? 0.193 Observation : DNA stock = 1mg/ml Table : OD reading at 595nm taken for different known standards and unknown test samples 2 1 3 BLANK STANDARD 1 STANDARD 4 The color intensity, is directly proportional to the DNA concentration.

16. Obtain the equation y = mx + c from the above data. Calculate the values of x, i.e., unknown sample. DNA Conc = Abs test- Abs blank/ Abs stand-Abs blank * conc of stand* dilution factor Or Calculation: Determine the amount of DNA in the unknown sample by plotting a standard curve of Absorbance 595nm on Y-axis and µg of DNA on X-axis. Assignment: Using the provided observation table, calculate the total DNA present in the given sample. What are the Advatanges and limiation of diphenylamine test?

17. Estimation of RNA Using Orcinol Method

18. Principle : The orcinol reaction, modified by Schneider, is the most sensitive colorimetric method for RNA estimation. RNA is depurinated in concentrated HCl, producing ribose phosphates which are dephosphorylated and dehydrated to form furfural. Furfural reacts with orcinol and Fe3+, yielding colored products with an absorption maximum at 660 nm. The color intensity, measured at 665 nm, is directly proportional to the RNA concentration. SOURCE: https ://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.himedialabs.com%2Feu%2Fcoasdstds%2Findex%2Fdownload%2Fid%2FHTBC007%2Fsource%2Ftds%2Flang%2FEN&psig=AOvVaw0bZoO_HaoUQ87U-mIEe0Ca&ust=1723059702424000&source=images&cd=vfe&opi=89978449&ved=0CBUQ3YkBahcKEwjo-sDmj-GHAxUAAAAAHQAAAAAQBA

19. Reagents : Modified Orcinol acids reagent: 6 % Orcinol and 10% FeCl3.6H20 in conc. HCl Standard RNA solution: 50mg RNA is dissolved in 50ml of 0.1N HCl (500ug/ml) RNA samples Tube Stock std ( ml of 1mg/ml ) Distilled water Extract (ml) 6% Orcinol (ml) Ferric chloride in conc.HCL Mix vigorously and keep in boiling water bath for 20 min. Cool to room temp and measure absorbance at 665nm blank 2 0.2 3 S1 0.1 0.9 0.2 3 S2 0.2 0.8 0.2 3 S3 0.4 0.6 0.2 3 S4 0.6 0.4 0.2 3 S5 0.8 0.2 0.2 3 T1 0.5 0.5 0.2 3 T2 1 0.2 3

20. Protocol Prepare a series of standard RNA dilutions in water to achieve concentrations of 100-800 µg/ml as shown in the table. Add 0.2 ml of 6% Orcinol and 3ml of Ferric chloride to each dilution, blank, standard, and unknown sample, and mix well. Use Tube B as the blank and Tubes S1 through S5 for the standard calibration curve. Use Tubes Test 1-2 for unknown samples. Mix them vigorously and Incubate all tubes in boiling water for 20 minutes. Cool the tubes and measure the absorbance at 665 nm against the blank. Construct a standard curve of absorbance (A665) vs. quantity of RNA. Calculate the concentration of the unknown DNA solution using the standard curve.

21. Preparation of reagents Preparation of standards Incubation Absorbance reading at 665nm After Incubation Pictorial representation of the Protocol :

22. Tube Stock std ( ml of 1mg/ml ) Absorbance at 665nm blank 0.018 S1 0.1 0.177 S2 0.2 0.266 S3 0.4 0.451 S4 0.6 0.659 S5 0.8 0.889 T1 ? 0.285 T2 ? 0.415 Observation RNA stock = 1mg/ml Table : OD reading at 665nm taken for different known standards and unknown test samples BLANK STANDARD 1 STANDARD 4 The color intensity, is directly proportional to the RNA concentration.

23. Calculations: Determine the amount of RNA in the unknown sample by plotting a standard curve of Absorbance 665nm on Y-axis and µg of RNA on X-axis. . RNA Conc = Abs test- Abs blank/ Abs stand-Abs blank * conc of stand* dilution factor RNA concentration in unknown sample can be calculated using the following formula: Obtain the equation y = mx + c from the above data. Calculate the values of x, i.e., unknown sample. Or Assignment: Using the provided observation table, calculate the total RNA present in the given sample . What are the advantage and limitation of Orcinol method?

24. References Division of Biochemistry. (2008). A practical manual of analytical biochemistry . Indian Veterinary Research Institute . Sambrook J, Russell D (2001) Molecular Cloning: A Laboratory Manual, 3rd edn . Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press . Plummer , D.T. (1977) An Introduction to Practical Biochemistry. Tata McGraw Hill, Bombay. https://d1vffmuvmgkypt.cloudfront.net/pdf/ridacom_ltd/himedia_laboratories/TD_HTBC006_pdf