Dna isolation Principle

Principle behind DNA isolation and roles of reagents used in isolation

1. DNA & PLASMID ISOLATION VPB-321: Animal Biotechnology VPB 321

2. BASICS What is DNA? What is the DNA made up of? What are the chemical properties of DNA? Where is the DNA located? VPB 321

3. WHAT IS DNA? Deoxy ribo nucleic Acid T he blueprint for the life . It is a molecule that encodes the genetic instructions used in the development and functioning of all known living organisms . DNA is a polymer or string of nucleotides . N ucleotide is composed of a sugar, a nitrogen base and a phosphate backbone. VPB 321

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5. WHAT IS THE DNA MADE UP OF? Sugar Moiety VPB 321

6. BONDS & BONDS VPB 321

7. WHAT ARE THE CHEMICAL PROPERTIES OF DNA? VPB 321

8. Stacking interactions Charge repulsion Charge repulsion The forces affecting the stability of the DNA double helix includes: Hydrophobic interactions - at outside and inside helps in stabilizing structure. Stacking interactions - these vander Waals forces are weak yet stabilizes the double helix. Hydrogen bonding - Faciliates stacking. Electrostatic interactions - DESTABILIZES This electrostatic interaction is primarily due to negative phosphates. It affects intrastrand and interstand inetractions. But can be neutralized with positive charges e.g., Na+ ions or proteins. CHEMISTRY OF DNA VPB 321

9. Where is/are the DNA located? Nucleus Mitochondria Plastids Bacterial nucleoid Plasmids Plasmids VPB 321

10. . Genomic DNA (SINGLE COPY) Plasmid DNA (MULTIPLE COPY) 1. Genomic DNA is chromosomal DNA where the genetic material is present. 1. Plasmid DNA is extra-chromosomal DNA in bacteria and some yeasts, i.e. Plasmid. 2. It is primary DNA in all living organisms. 2. It is secondary DNA. 3. It is linear in eukaryotes whereas circular in prokaryotes 3. It is circular. 4. As it encodes genetic information, it is much larger than plasmid. 4. It is smaller. 5. Genomic DNA is organized with proteins called histones. 5. Plasmid DNA is not with histones. 6.  It contains essential genes which codes for functional and structural proteins. 6. It contains non-essential genes. 7. It can be transferred only through cell division within the same species. 7. It can be transferred through horizontal way of gene transfer between same or different species. VPB 321

11. HOW TO TAKE THIS DNA OUT OF THE CELL? VPB 321

12. DNA ISOLATION vs DNA EXTRACTION DNA ISOLATION Isolation aims to get as much of the target out of your sample as possible. Along the step of isolation one usually picks up quite a bit of contamination. DNA EXTRACTION Extraction is just one specific way to achieve isolation and purification. Extraction uses a solvent that serves as extractant and can be devided in above stages: 1) Gentle lysis of the cells / solubilization of DNA or isolation 2) Removal of contaminants (proteins, RNA and other macromolecules) or the so-called purification is achieved either by enzymatic or chemical means. https://www.researchgate.net/ VPB 321

13. DIFFERENT SOURCES FOR DNA ISOLATION DNA can be isolated from all the types living cells and fossilsized cells containing nucleus or nucleiod or semi autonomous organelles. The viral DNA can also be isolated. For example: Blood, other body fluids, bacterial culture etc. We will overview: Genomic DNA isolation from blood, plasmid DNA isolation and bacterial genomic DNA isolation VPB 321

14. Genomic DNA Extraction SOURCE: MICROBES & BLOOD VPB 321

15. BASIC DIFFERENCES Genomic DNA (gDNA) extraction is the simpler procedure because strong lysis is the only step necessary to release gDNA into solution. For yeast, plants and bacteria, lysis involves enzymatically breaking the strong, rigid cell wall before mechanically disrupting the plasma membrane. VPB 321

16. STEPS STEPS: 1. Lysis or cells disruption 2. Phase seperation 3. Clearing proteins 4. Precipitating DNA VPB 321

17. 1. Lysis or cells disruption Extraction buffer and lysis buffer and incubation at 65°C. These extraction/ lysis buffer contains a high concentration of chaotropic salts. Firstly, they destabilize hydrogen bonds, van der Waals forces and hydrophobic interactions, leading to destabilization of proteins, including nucleases. Secondly, they disrupt the association of nucleic acids with water. NaCl : phosphate of DNA molecule repel one molecule from others. Na+ ions form an ionic bond with phosphates and neutralized the negative charge allowing DNA molecules grouping. EDTA (Ethylenediamine tetraceticacid): chelating agent with high affinity to metallic ions of Mg, DNAse cofactors (enzymes that degrade the DNA). EDTA bind to ions and overturn its effects. CTAB (Hexadecyl trimethyl-ammonium bromide): detergent used to break cellular membranes and remove lipids Other stabilizer agents : Protinase-K, Tris HCl, sorbitol, sodium bisulphite, DTT, detergents: SDS (remove lipids), sarkosyl, triton, PVP (bind to polyphenols- components of vegetable cellular wall-removing), 2-mercaptoethanol (denature proteins). VPB 321

18. GRAM NEGATIVE VS GRAM POSITIVE - LYSIS The presence of a high peptidoglycan content in the cell walls of Gram positive bacteria is a major hurdle in the isolation of DNA. So it’s resistance to conventional methods of lysis . Lysozyme + detergents such as SDS ensures better lysis of the Gram positive cell wall . Penicillins* which is known for interfering in the assembly of N-Acetylglucosamine (NAG) and N-Acetylmuramic acid (NAM) moieties during Gram positive cell wall synthesis can also solve the purpose. *De, Sachinandan et al. “A Simple Method for the Efficient Isolation of Genomic DNA from Lactobacilli Isolated from Traditional Indian Fermented Milk (Dahi).” Indian Journal of Microbiology 50.4 (2010): 412–418. PubMed Central. Web. VPB 321

19. BLOOD DNA ISOLATION -LYSIS Enzymes may also feature LYSIS of RBC and WBC in presence of lysisbuffer. The broad-spectrum serine protease proteinase K is also very efficient in digesting proteins away from nucleic acid preparations. VPB 321

20. 2. PHASE SEPARATION Addition of chloroform-isoamylalcohol (24:1) or phenol- chloroform isoamylalcohol (25:24:1). Centrifugation. Organic solvents, hydrophobics lysates keep trapped, eg. membrane lipids, proteins or polysacharids. Besides denature proteins. VPB 321

21. ROLE OF PHENOL DNA is negatively charged molecule, therefore soluble in polar solvent like water. Whereas proteins has both polar (hydrophilic) and non polar (hydrophobic) side chains amino acids. In cell normally the DNA remain dissolved in polar phase and the proteins tend to fold in such a way that their hydrophilic or polar groups face outward in solvent and the hydrophobic toward inside. On adding phenol, proteins flip since phenol is less polar than water. Now, therefore protein (also lipids) becomes soluble in phenol or non polar phase and DNA in water phase. In such a way we acheive separation of DNA from proteins (lipids). VPB 321

22. ROLE OF PHENOL & ISOAMYL ALCOHOL Chloroform is significantly denser than water, so adding it to the organic phase increases the overall density of that phase, helping to prevent phase inversion. Sometimes, the choloroform when added next to the P:C treatment, helps in extracting phenol from the aqueous solution Isoamyl alcohol acts as anti-foaming agent. VPB 321

23. 3. CLEARING OUT PROTEINS Proteins are eliminated adding a protease and increasing the osmolarity (sodium acetate or ammonium acetate) DNA precipitates with alcohol – usually pure and could ethanol or isopropanol (2-propanol). Because DNA is non-soluble in alcohol, precipitate and form a pellet in the bottom of the tube after centrifugation. This step also remove alcohol soluble salts. DNA cleans with 70% ethanol, dry and dilute in TE buffer (protect DNA from degradation) or sterile distilled water. 4. PRECIPITATING DNA VPB 321

24. BLOOD DNA ISOLATION PROTOCOL GENOMIC DNA ISOLATION FROM HUMAN WHOLE BLOOD SAMPLES BY NON ENZYMATIC SALTING OUT METHOD. SUGUNA et. al. International Journal of Pharmacy and Pharmaceutical Sciences. Vol 6, Issue 6, 2014 VPB 321

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26. MICROBIAL DNA ISOLATION PROTOCOL He, Fanglian. “E. Coli Genomic DNA Extraction.” BIO-PROTOCOL 1.14 (2011): n. pag. CrossRef. Web. 8 Apr. 2017. VPB 321

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29. PLASMID DNA VPB 321

30. STEPS Plasmid DNA extraction is a bit trickier because plasmid DNA must be kept separate from gDNA. This separation is based on size, and good separation relies on using the right lysis method. STEPS: 1.  ALKALINE Lysis or cells disruption 2. Phase seperation 3. Clearing proteins 4. Precipitating DNA VPB 321

31. ALKALINE LYSIS? For plasmid DNA extraction, the lysis has to be a lot more subtle. Firstly perform a SDS bases lysis as done in case of gDNA. Next the sample is neutralized in a potassium acetate solution to renature the plasmid, and this is key to the separation of plasmid and gDNA. Because plasmids are small, they can easily reanneal forming dsDNA. Genomic DNA, however, is too long to reanneal fully, and instead it tends to tangle so that complimentary strands remain separated. During centrifugation, gDNA (bound to protein) forms a pellet while plasmid DNA remains soluble. It is key at this step not to vortex or mix the sample vigorously because gDNA breaks easily, and broken gDNA may be small enough to reanneal and go into solution with the plasmid. VPB 321

32. PLAMSID DNA ISOLATION PROTOCOL He, Fanglian. “Plasmid DNA Extraction from E. Coli Using Alkaline Lysis Method.” BIO-PROTOCOL 1.1 (2011): n. pag. CrossRef. Web. 8 Apr . 2017. VPB 321

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34. DNA QUANTIFICATION HOW TO CHECK DNA THAT YOU EXTRACTED? VPB 321

35. 1. Electrophoresis in agarose gels- qualitative VPB 321

36. 2. Espectrophotometer/ nanometer- quantitative Gives the 260/ 280 ratio which tells us about purity of DNA. 260/280 = 1.8 stable 260/280 = 2.0 > (contaminated with proteins) 260/280 = 1.6 < (contaminated with RNA) Also it quantifies and displays concentration of DNA in ng/ul. VPB 321

37. THANK YOU QUERIES? VPB 321