Molecular and Biochemical Techniques PDF

Analyzing nucleic acids – DNA and RNA Successful nucleic acid purification required four important steps: 1.effective disruption of cells or tissue; 2.denaturation of nucleoprotein complexes; 3.inactivation of nucleases, for example, 4.RNase for RNA extraction and DNase for DNA extracti...

Analyzing nucleic acids – DNA and RNA Successful nucleic acid purification required four important steps: 1.effective disruption of cells or tissue; 2.denaturation of nucleoprotein complexes; 3.inactivation of nucleases, for example, 4.RNase for RNA extraction and DNase for DNA extraction Type of Nucleic Acid Extraction Conventional Method 1. Guanidinium Thiocyanate-Phenol-Chloroform Extraction using phenol: chloroform: isoamyl alcohol (25:24:1) ü Proteins, lipids, carbohydrates, and cell debris are removed through extraction of the aqueous phase with the organic mixture of phenol and chloroform. ü Guanidinium thiocyanate is a chaotropic agent used in protein degradation. (A biphasic emulsion forms when phenol and chloroform are added. The hydrophobic layer of the emulsion will then be settled on the bottom and the hydrophilic layer on top by centrifugation. The upper phase which contained DNA is collected and DNA can be precipitated from the supernatant by adding ethanol or isopropanol in 2:1 or 1:1 ratios and high DNA extraction with concentration of salt. DNA precipitate is collected by phenol/chloroform/isoamyl alcohol centrifugation, and excess salt is rinsed with 70% ethanol and pH 8 - aqueous top phase contains centrifuged to discard the ethanol supernatant. The DNA the majority of DNA, interphase pellet is then dissolved with TE buffer or sterile distilled water) mostly proteins, and lower organic phase most of the RNA and lipids In the acidic conditions, total RNA will remain in the upper aqueous phase of the whole mixture, while DNA and proteins remain in the interphase or lower organic phase. Recovery of total RNA is then done by precipitation with isopropanol 2. Alkaline Extraction Method: https://www.youtube.com/watch?v=O4oLyd2mZv8 The principle of the method is based on selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double stranded. Bacterial proteins, broken cell walls, and denatured chromosomal DNA enmeshed into large complexes that are coated with dodecyl sulfate. Plasmid DNA can be recovered from the supernatant after the denatured material has been removed by centrifugation. 3. CTAB Extraction Method. For plant extraction, the initial step that needs to be done is to grind the sample after freezing it with liquid nitrogen. The purpose of doing this step is to break down cell wall material of sample and allow access to nucleic acid while harmful cellular enzymes and chemicals remain inactivated. After grinding the sample, it can be resuspended in a suitable buffer such as CTAB. Cetyltrimethylammonium bromide (CTAB) is a nonionic detergent that can precipitate nucleic acids and acidic polysaccharides from low ionic strength solutions. Meanwhile, proteins and neutral polysaccharides remain in solution under these conditions. In solutions of high ionic strength, CTAB will not precipitate nucleic acids and forms complexes with proteins. CTAB is therefore useful for purification of nucleic acid from organisms which produce large quantities of polysaccharides such as plants and certain Gram-negative bacteria  Spectrophotometry https://www.youtube.com/watch?v=pxC6F7bK8CU https://www.ncbionetwork.org/iet/spectrophotometer/ Concepts Transmittance Absorbance Extinction coefficient - The extinction coefficient is a characteristic that determines how strongly a species absorbs or reflects radiation or light at a particular wavelength. It is an intrinsic property of the isolates that is dependent on the atomic, chemical, and protein structural composition of the isolate sequence The Beer-Lambert law states that there is a linear relationship between the concentration and the absorbance of the solution, which enables the concentration of a solution to be calculated by measuring its absorbance. https://www.edinst.com/blog/the-beer-lambert-law/  Agarose gel electrophoresis ü Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by hydrogen-bonding when heated in a buffer and allowed to cool ü The most popular medium for the separation of moderate and large-sized nucleic acids and have a wide range of separation ü The concentration of agarose is referred to as a percentage of agarose to volume of buffer (w/v), and agarose gels are normally in the range of 0.2% to 3%. ü In general, if the aim is to separate large DNA fragments, a low concentration of agarose should be used, and if the aim is to separate small DNA fragments, a high concentration of agarose is recommended. ü The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes like https://www.youtube.com/watch? bromophenol blue and xylene cyanol dyes. v=saJIWFUGEbw ü Role of ethidium bromide  Protein purification https://www.youtube.com/watch?v=PVvpEKeOzEM The material till affinity purification is important for the lecture exam  SDS-PAGE gel electrophoresis Separation of proteins based on charge/mass (e/m) ratio https://www.youtube.com/watch?v=i_6y6Z5UvwE Role of key terms Concepts: SDS beta-mercaptoethanol or DTT Buffers Heating Electrodes Ammonium persulfate Stacking and Separating gel – pH? TEMED Acrylamide Chloride ions Concentration of gels Glycine Molecular weight markers Bromophenol blue Glycerol or sucrose  Chromatography https://www.youtube.com/watch?v=0m8bWKHmRMM Gel filtration Sephadex G 50 https://www.youtube.com/watch?v=S89mAyh6yHU Ion exchange https://www.youtube.com/watch?v=lp40a7mtc4E Affinity chromatography https://www.youtube.com/watch?v=ezj9MVGCf8c