Nucleic Acid Extraction & Electrophoresis & Quantification

1. Nucleic Acid Extraction & Electrophoresis & Quantification By Dr. Emad AbdElhameed Morad Lecturer of Medical Microbiology and Immunology

2. DNA is easy to prepare and store because DNAases are easy destroyed by heating. • RNA preparation is difficult because RNAases are extremely stable. • In this lecture we will talk about: • Nucleic acid extraction: • DNA • Plasmid • RNA • Electrophoresis. • Quantification of the extracted nucleic acid.

3. DNA extraction • Phenol/chloroform/isoamyl alcohol extraction: • Phenol is organic solvent. • Nucleic acids prefer aqueous solutions. • But, proteins, lipids and carbohydrates prefer organic environment. • So, after addition of phenol to the cell lysate, the reaction tube will have two phases: • Upper aqueous layer containing DNA. • Lower organic layer containing phenol + proteins.

4. Phase separation by phenol extraction

5. Salting out method: • Cell lysis. • Protein digestion by proteinase enzyme. • Protein precipitation by high salt concentration. • Salt also reduces the repulsion of the negatively charged DNA molecules. • Centrifugation will remove the precipitated proteins. • The supernatant contains the DNA. • DNA is then precipitated by adding ethanol. • The precipitated DNA is resuspended in the desired buffer.

6. Spin column: • Commercial DNA extraction kits are based on selective adsorption of DNA to silica gel membrane columns or glass fiber matrix columns. • Binding of DNA to silica or glass fiber is enhanced by high salt concentration. • The bound DNA will then be eluted by elution buffer with low salt concentration.

7. Extraction by spin column

8. Plasmid extraction • Alkaline lysis method: • Cell lysis. • Then, we add sodium hydroxide. • The alkaline pH will denaturate chromosomal DNA but not the plasmid DNA. • Then we add sodium or potassium acetate that will neutralize pH. • So, chromosomal DNA will renature and aggregate with proteins. • By centrifugation, proteins + chromosomal DNA will precipitate while plasmid DNA will remain in the supernatant.

9. RNA extraction • Successful RNA isolation depends on: • Suppression of endogenous RNAases. • Avoid contamination with exogenous RNAases during extraction. • Samples should be processed immediately or stored at -70 degree until required. • Inactivation of RNAases by strong denaturing agents like urea, guanidinium hydrochloride, guanidinium isothiocyanate. Suppression of endogenous RNAases

10. Avoid contamination with exogenous RNAases during extraction • Specify glassware, solutions, equipments to be used for RNA extraction only. • Treat water and laboratory utensils with diethylpyrocarbonate (DEPC) which is a strong RNAase inhibitor. DEPC is a suspected carcinogen. • Autoclave glassware, solutions and equipments if possible. • Use disposable gloves, disposable plastic materials that must be RNAase free.

11. Methods of RNA extraction • Guanidinium isothiocyanate extraction: • Cell lysis. • Protein denaturation by guanidinium isothiocyanate. • Cell lysate is mixed with cesium chloride. • The density of RNA in cesium chloride is much greater than of other cellular elements. • During ultracentrifugation, RNA pellets at the bottom of the tube and becomes separated from other cellular components.

12. RNA extraction by Trizol: • Tizol is a monophasic solution of phenol & chloroform + guanidinium isothiocyanate. • The presence of phenol & chloroform will separate cell lysate into two layers: • Upper aqueous layer containing RNA. • Organic layer containing proteins. • RNA is then precipitated from the aqueous layer by isopropyl alcohol. • RNA extraction by spin column: • These columns use RNA adsorbing silica or glass fiber. • RNA is then eluted by elution buffer.

13. RNA extraction by Trizol

14. RNA extraction by magnetic separation technology: • Couple magnetic beads to silica. • Magnetic silica beads binds RNA in the lysate. • The conjugated magnetic beads are then collected by applying magnetic field. • RNA is then eluted from the beads.

15. Electrophoresis • DNA and RNA carry negative charges. • After extraction, they are subjected to horizontal electrophoresis in agarose gel. • Ethidium bromide or SYBR green dyes are added to the gel. • Ethidium bromide inserts itself between the nucleotides of DNA or RNA and fluoresce under ultraviolet light.

16. Genomic DNA is extremely long. So, it must be cut by restriction endonuclease enzyme. • The resulting DNA fragments move toward positive electrode according to their sizes producing bands. • Pulsed field gel electrophoresis (PFGE)is used to separate large pieces of DNA

17. Quantification • Quantification of extracted nucleic acids is done by using spectrophotometer as follows: • At wave length 260: optical density (OD) of 1 means that: • The concentration of DNA= 50 µg/ml • The concentration of RNA= 40 µg/ml • So, the concentration of extracted DNA in a sample= OD at 260 x 50 x dilution factor

18. Spectrophotometer

19. It is very important to assess purity of the extracted DNA (the degree of protein contamination). • Purity is assessed by: • Determine the ratio between the OD of the sample at 260 nm and 280 nm. (OD 260/OD 280). • If the ratio is 2: this means that protein contamination is zero. • If the ratio is < 2:this means protein contamination of the extracted DNA. N.B. Notice that the concentration of the extracted nucleic acid in a given sample can be roughly estimated by observing the fluorescence intensity of the band obtained on agarose gel after electrophoresis.

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