Molecular Biology Lab - 03 DNA Quantitation

MOLECULAR BIOLOGY AND DIAGNOSTICS LABORATORY 8. DNA QUANTITATION 9. Check quality of sample by taking ratio of A230/260/280, A260/280 and A260/230. Calculate quantity of sample as per equation and note it. RESULTS OF THE TEST OUTLINE • DNA Quantitation (Canvas) o Introduction o Principle of the Test o Materials o Procedure o Results of the Test • DNA Quantitation o Spectrophotometric Analysis of DNA o Fluorometric Spectroscopy o Electrophoresis DNA QUANTITATION (CANVAS) Wavelength Characteristic (nm) nature 215-230 Minimum absorbance for nucleic acids. Peptide bonds in proteins absorb light INTRODUCTION • Quantification and assessment of DNA/RNA and Protein purity and concentration, is first entry step in most of molecular biology protocol routinely employed in many lab. DNA Quantitation is commonly performed to determine the average concentrations of DNA or RNA present in a mixture, as well as their purity. There are two main approaches used by scientists to quantitate, or establish the concentration, of nucleic acids (such as DNA or RNA) in a solution. These are spectrophotometric quantification and UV fluorescence tagging in presence of a DNA dye. 260 PRINCIPLE OF THE TEST • • Spectrophotometric analysis is based on the principles that nucleic acids absorb ultraviolet light in a specific pattern. In the case of DNA and RNA, a sample is exposed to ultraviolet light at a wavelength of 260 nanometers (nm) and a photo-detector measures the light that passes through the sample. Some of the ultraviolet light will pass through and some will be absorbed by the DNA / RNA. The more light absorbed by the sample, the higher the nucleic acid concentration in the sample. Checking quality of sample for detection of Protein, or other contaminant with DNA/RNA sample is most common practice. For standard practice for DNA sample usually A260/A280 are used, as if ratio is 1.8 indicate good pure quality of DNA, while ratio of 2.0 indicate contamination of RNA in sample and same ratio less than or 0.6 is indicating protein in sample. MATERIALS • • DNA/RNA Sample Buffer 270 280 330 or higher Remarks Measurements are generally not performed at this wavelength because commonly used buffers and solvents, such as Tris, also absorb at these wavelengths. Contaminant phenolate ion, thiocyanate and other organic compound absorb at this range. Nucleic acid has Purine’s absorbance maximum maximum is slightly below absorbance at this 260; pyrimidines maximum, wavelength is slightly above 260. Purines have a higher molar absorptivity than pyrimidines. Therefore, the absorbance maximum and absorptivity of a segment of DNA depends on its base composition. Phenol absorbs Phenol may be strongly contamination Protein absorbance Aromatic amino acid peak strongly absorbs at 280 Nm Non protein and Presence of particulate Nucleic acid matter causing light absorbance range scattering DNA QUANTITATION SPECTROPHOTOMETRIC ANALYSIS OF DNA • • • • Principle: Nucleic acids absorb ultraviolet light in a specific pattern. Nucleic acids absorb light at 260 nm Absorptivity constants = (50 for DNA, 40 for RNA) Concentration to Absorbance: PROCEDURE 1. 2. 3. 4. 5. 6. 7. Start on spectrophotometer and adjust it to wavelength 260 nM. Auto-zero spectrum with blank of TE buffer for DNA/RNA or PBS buffer Protein sample. Withdraw buffer and rinse cuvette once with distilled water. Fill cuvette with max volume with DNA/RNA/Protein sample. Run absorbance spectrum in range of 230 to 330 Nm. Run Peak value and find point with maximum absorbance in range of scan and note it Further, take absorbance at 230, 260 and 280 Nm and note it. • • One optical density unit (or absorbance unit) at 260 nm = 50 mg/L (or 50 g/mL) of DNA and 40 g/mL of RNA Example 1: A DNA preparation diluted 1:100 yields an absorbance reading of 0.200 at 260 nm. To obtain the concentration in μg/mL, multiply: 0.200 absorbance units x 50 μg/mL per absorbance unit x100= 1000 μg/mL o If in this case, the DNA was eluted or resuspended in a volume of 0.5mL,the yield would be: 1000 mL x 0.5 mL= 500 μg • Example 2: An RNA preparation diluted 1:10 yields an absorbance reading of 0.500 at 260 nm. The concentrations is: 0.500 absorbance units x 40 μg/mL per absorbance unit x10= 200 μg/mL o If in this case, the RNA was eluted or resuspended in a volume of 0.2 mL,the yield would be: 200 μg/mL x 0.2 mL= 40 μg Wavelength Characteristic (nm) nature 215-230 Minimum absorbance for nucleic acids. Peptide bonds in proteins absorb light 260 Nucleic acid has maximum absorbance at this wavelength 270 280 330 or higher Phenol absorbs strongly Protein absorbance peak Non protein and Nucleic acid absorbance range GEL SYSTEMS • • • • Remarks Measurements are generally not performed at this wavelength because commonly used buffers and solvents, such as Tris, also absorb at these wavelengths. Contaminant phenolate ion, thiocyanate and other organic compound absorb at this range. Purine’s absorbance maximum is slightly below 260; pyrimidines maximum, is slightly above 260. Purines have a higher molar absorptivity than pyrimidines. Therefore, the absorbance maximum and absorptivity of a segment of DNA depends on its base composition. Phenol may be contamination • Provide resistance to the movement of molecules Prevent diffusion and reduce convection currents Serve as a support medium o Agarose gels o Polyacrylamide gel Agarose Gels o Extracted from seaweed o Component of agar o Linear polymer of agarobiose Polyacrylamide Gel o Originally used mostly for Protein separation o Used for sequencing used for sequencing nucleic acids, mutation analyses, nuclease protection assays o Advantages ▪ Higher resolution capability for small fragments ▪ The components of polyacrylamide gels are synthetic Aromatic amino acid strongly absorbs at 280 Nm Presence of particulate matter causing light scattering FLUOROMETRY/FLOUROSCENT SPECTROSCOPY • • • • • • Measures fluorescence related to DNA concentration in association with DNA specific fluorescent dyes Early method uses DABA Modern method uses DNA specific dye (Hoechst 33258) PicoGreen - more sensitive OliGreen - single stranded DNA SyBr Green II - RNA stain ELECTROPHORESIS • • • BUFFER SYSTEMS • • • • Movement of molecules by an electric current Done in a matrix to limit migration and contain the migrating material Protein and Nucleic acids To carry current and protect samples during electrophoresis Solution of a weak acid and its conjugate base Commonly used: o Tris borate EDTA (TBE) o Tris phosphate EDTA (TPE) o Tris acetate EDTA (TAE) Advantages and disadvantages: o TBE has a greater buffering capacity than TAE o TBE is not recommended for some postelectrophoretic isolation procedures. o When using any buffer, care must be taken that the gel does not overheat o Stock solutions of TBE are prone to precipitation NUCLEIC ACID SPECIFIC DYES • • Ethidium bromide o Emit orange light (300nm) o Care must be taken (carcinogenic) SyBr green o Also emits light in the orange range o 25–100 times more sensitive than EtBr o preferred dye for real-time PCR methods • Silver Stain o More sensitive staining system o Sample is fixed with methanol and acetic acid o Two procedures: silver diamine and silver nitrate ELECTROPHORESIS EQUIPMENT •