DNA and RNA extraction

DNA and RNA extraction

1. DNA and RNA Extraction Prof. GHIZAL FATIMA ERA UNIVERSITY, LUCKNOW

2. DNA isolation  is a process of  purification  of  DNA  from sample using a combination of physical and chemical methods. The first  isolation   of DNA  was done in 1869 by Friedrich Miescher . Currently it is a routine procedure in molecular biology or forensic analyses . For the chemical method, there are many different kits used for extraction, and selecting the correct one will save time on kit optimization and extraction procedures. PCR sensitivity detection is considered to show the variation between the commercial kits

3. BASIC PROCEDURE There are three basic and two optional steps in a DNA extraction : Cells which are to be studied need to be collected. Breaking the cell membranes open to expose the DNA along with the cytoplasm within (cell lysis ). Lipids from the cell membrane and the nucleus are broken down with detergents and surfactants. Breaking proteins by adding a protease (optional). Breaking RNA by adding an  RNase  (optional). The solution is treated with concentrated salt solution to make debris such as broken proteins, lipids and RNA to clump together. Centrifugation of the solution, which separates the clumped cellular debris from the DNA.

4. DNA purification from detergents, proteins, salts and reagents used during cell lysis step. The most commonly used procedures are: Ethanol precipitation usually by ice-cold ethanol or  isopropanol . Since DNA is insoluble in these alcohols, it will aggregate together, giving a  pellet  upon centrifugation. Precipitation of DNA is improved by increasing of ionic strength, usually by adding sodium acetate. Phenol–chloroform extraction  in w ich  phenol denatures proteins in the sample. After centrifugation of the sample, denaturated proteins stay in the organic phase while aqueous phase containing nucleic acid is mixed with the chloroform that removes phenol residues from solution. Minicolumn purification  that relies on the fact that the nucleic acids may bind (adsorption) to the solid phase (silica or other) depending on the pH and the salt concentration of the buffer.

5. Cellular and  histone  proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation . After isolation, the DNA is dissolved in slightly alkaline buffer, usually in the TE buffer ( The  TE Buffer is  used for  protection  of  DNA and RNA from degradation. The Tris buffering agent and EDTA metal chelating properties help  protect DNA  and RNA), or in ultra-pure water.

6. Special types Specific techniques must be chosen for isolation of DNA from some samples. Typical samples with complicated DNA isolation are: archaeological samples containing partially degraded DNA. samples containing inhibitors of subsequent analysis procedures, most notably inhibitors of PCR, such as  humic acid from soil, indigo and other fabric dyes or  haemoglobin  in blood samples from microorganisms with thick cellular wall, for example yeast

7. Extrachromosomal DNA ( DNA  that is found outside the nucleus of a cell. It is also referred to as extranuclear DNA  or cytoplasmic   DNA . Most  DNA  in an individual genome is found in chromosomes but  DNA  found outside the nucleus also serves important biological functions) is generally easy to isolate, especially plasmids may be easily isolated by cell lysis followed by precipitation of proteins, which traps chromosomal DNA in insoluble fraction and after centrifugation, plasmid DNA can be purified from soluble fraction. A Hirt DNA Extraction is an isolation of all  extrachromosomal DNA in a mammalian cell. The Hirt extraction process gets rid of the high molecular weight nuclear DNA, leaving only low molecular weight mitochondrial DNA and any viral  episomes  present in the cell.

8. Detecting DNA A diphenylamine (DPA) indicator confirms the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95 °C) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to w- hydroxylevulinyl aldehyde , which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations.

9. Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nms , and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has a ratio of 1.8 at 260/280 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.

10. DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose  gel, staining with  ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration . Using the Southern blot technique, this quantified DNA can be isolated and examined further using PCR and  (Restriction Fragment Length Polymorphism)   RFLP  analysis . These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which forensic  scientists and molecular biologist use for comparison, identification, and analysis.

11. Q1-What are the three main steps of DNA extraction? A1-Step 1: Lysis . In this step, the cell and the nucleus are broken open to release the DNA inside and there are two ways to do this. ... Step 2: Precipitation . Ethanol precipitation is a commonly used technique for concentrating and de-salting nucleic acids (DNA or RNA) preparations in aqueous solution. The basic procedure is that salt and ethanol are added to the aqueous solution, which forces the precipitation of nucleic acid out of solution. Step 3: Purification . Q2- Why is DNA extracted from human cells? DNA  is extracted from human cells for a variety of reasons. With a pure sample of DNA you can test a newborn for a genetic disease, analyze forensic evidence, or study a gene involved in cancer etc.

12. Q3-What is the function of sodium dodecyl sulphate in a DNA extraction? A3-Sodium Dodecyl Sulfate, is a detergent that is known to denature proteins. ... It is also used in nucleic acid extraction procedures for the disruption of cell walls and dissociation of nucleic acid:protein complexes. Q4-Can DNA be extracted from urine? Urine is not considered an ideal source of DNA due to the low concentration of nucleated cells present in human urine. The nucleated cells found in urine are typically white blood cells and epithelial cells . Q5-What is the importance of DNA extraction? A-5-  S tudying the genetic causes of disease and for the development of diagnostics and drugs. It is also essential for carrying out forensic science, sequencing genomes, detecting bacteria and viruses in the environment and for determining paternity . Q6-Why do you use alcohol in DNA extraction? A6-Since  DNA  is insoluble in  ethanol  and isopropanol , the addition of  alcohol , followed by centrifugation, cause the  DNA  proteins to come out of the solution. When DNA  concentration in the sample is heavy, the addition of  ethanol  will cause a white precipitate to form immediately.

13. RNA EXTRACTION RNA extraction  is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of  ribonuclease  enzymes in cells and tissues, which can rapidly degrade RNA .  Several methods are used in molecular biology to isolate RNA from samples, the most common of these is  Guanidinium thiocyanate -phenol-chloroform extraction. The filter paper based lysis and elution method features high throughput capacity . RNA extraction in liquid nitrogen, commonly using a mortar and pestle (or specialized steel devices known as tissue pulverizers ) is also useful in preventing ribonuclease activity.

14. RNA (Ribonucleic acid) is a polymeric substance present in living cells and many viruses, consisting of a long single-stranded chain of phosphate and ribose units with the  nitrogen bases adenine, guanine, cytosine, and uracil , which are bonded to the ribose sugar. RNA is used in all the steps of protein synthesis in all living cells and carries the genetic information for many viruses .   The isolation of RNA with high quality is a crucial step required to perform various molecular biology experiment. TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues .

15. TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. Addition  of  chloroform, after the centrifugation, separates the solution  into  aqueous and organic phases. RNA  remains only in  the  aqueous  phase.

16. After transferring the aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol . But the DNA and proteins can recover by sequential separation after the removal of aqueous phase. Precipitation with ethanol requires DNA from the interphase , and an additional precipitation with isopropyl alcohol requires proteins from the organic phase. Total RNA extracted by TRIzol Reagent is free from the contamination of protein and DNA. This RNA can be used in Northern blot analysis, in vitro translation, poly (A) selection, RNase protection  assay, and molecular cloning.

17. Procedure Materials Required Reagents Chloroform (without any additives, such as isoamyl alcohol)  Isopropyl alcohol  75% Ethanol (in DEPC-treated water)   RNase -free water or 0.5% SDS solution The SDS solution must be prepared using DEPC-treated, autoclaved water.  DEPC  inactivates the RNases by the covalent modifications of the histidine residues.

18. Homogenization Growth medium on the cells was discarded and cells were washed with ice cold 1X PBS. The monolayer was then covered with 1 ml of l TRIzol and the cells were lysed and homogenized by repeated pipetting .

19. Phase Separation    The homogenized samples were incubated for 5 minutes at 15 to 30°C for the complete dissociation of nucleoprotein complexes . 0.2 ml (200 microliters )of chloroform per 0.75 ml of TRIZOL LS Reagent was added. The tubes were shaked vigorously by hand for 15 seconds and incubated them at 15 to 30°C for 2 minutes . The samples were centrifuged for 15 minutes at no more than 12,000 g (4°C ). The aqueous phase was transferred to other tubes. ( Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase , and a colorless upper aqueous phase. RNA remains only in the aqueous phase. The volume of the aqueous phase is about 70% of the volume of TRIZOL LS Reagent used for homogenization.)

20. RNA Precipitation The RNA was precipitated from the aqueous phase by mixing with 3 microlitre of glycogen and 500 microlitre of isopropyl alcohol. The mixture was centrifuged for 30 minutes at 12,000 × g (2 to 8°C).( The RNA precipitate forms a gel-like pellet on the side of the tube at bottom).

21. RNA Wash The supernatant was removed. The RNA pellet was washed once with 75% ethanol, adding 900 microlitre of 75% ethanol per 0.75 ml of TRIZOL LS Reagent used for the initial homogenization. The sample were inverted and mixed and centrifuged at 12,000 rpm for 30 minutes at 4 degree.

22. Redissolving RNA The RNA pellet was dried . RNA was dissolved in RNase -free water (or 0.5% SDS solution) by passing the solution through the pipette tip for a few times, and incubating for 10 minutes at 55 to 60°C.     Note : 0.5% SDS should not be used if RNA will undergo further enzymatic reaction. DNA can also be in 100% formamide ( deionized ) and stored at -70°C.

23. Precautions for preventing RNase contamination: RNases can be introduced into the RNA preparation at any point accidently during the isolation procedure through improper technique. RNase activity is difficult to inhibit, so it is very essential to prevent its introduction. The following guidelines should be observed carefully while working with RNA . Always wear disposable gloves. Usually our skin contains many bacteria and molds that can contaminate the RNA preparation and be a source of RNases . Good and clean microbiological techniques will prevent microbial contamination. Use sterile, disposable plastic ware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases from shared equipment.

24. Q1-Why trizol is used for RNA isolation? TRIzol  Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues.TRIzol  works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. ...RNA remains only in the aqueous phase . Q2-What is RNA extraction used for? This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA . Q3-What does chloroform do in RNA isolation? After solubilization , the addition of chloroform causes phase separation (much like extraction with phenol:chloroform:isoamyl alcohol), where protein is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueous phase.