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Nucleic Acid Purification

Nucleic Acids: Composition,Purpose of Purification,Modes of Purification & Latest Research Trends.

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Nucleic Acid Purification

  1. 1. Topic : Nucleic Acid Purification Presented by: D.Sairam Course: Bioanalytical and Instrumentation-I Course Code: BSBT-202 Course Instructor: Dr.Subhabrata Kar
  2. 2. Overview of the Presentation • Introduction ( Nucleic Acids) • Purpose of Purification • Methods of Nucleic Acid Purification • Research Trends in Nucleic Acid Purification • References
  3. 3. Introduction  Nucleic acids are polymolecules, or large biomolecules, essential for all known forms of life. Nucleic acids, which include DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid), are made from monomers known as nucleotides.  DNA purification is a technique that removes impurities and unused reagents from samples after enzymatic reactions, such as PCR.  There are three major large scale processes of DNA Extraction namely: • Spin Column-based Nucleic acid Purification • Boom method (Boom nucleic acid extraction method) • Phenol–chloroform extraction
  4. 4. Purpose of Nucleic Acid Purification • It usually determines the success or failure of all your immediate and downstream experimentation. • Extract ample amounts of your genomic and/or plasmid DNA sample from a limited source to satisfy the requirements of your research. • Purify it to reduce the amount of contaminants that can compromise the results of your research and shorten the shelf- life of your precious sample.
  5. 5. Boom Method (Boom Nucleic Acid Extraction Method)  This method is one of the most widespread method for isolating nucleic acids from biological samples and is known as a simple, rapid, and reliable method for the small-scale purification of NA from Biological sample.  The crux of the method of this method is the use of Silica beads, capable of binding the NA in the presence of a chaotropic substance.  This method is said to have been developed and invented by Willem R. Boom in 1990.  Amorphous silicon oxide and glass powder, Alkyl silica, Aluminium silicate (zeolite), or, activated silica with -NH2 (Amino) are suitable as nucleic acid binding solid phase material.  Today, the embodiments of Boom method, characterized by "utilizing the magnetic beads (silica beads are magnetic beads)" is widely used.
  6. 6. Spin Column-based Nucleic acid Purification  Genesis of the Process: DNA binds to silica, glass particles or to unicellular algae called diatoms which shield their cell walls with silica. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions.  It is a solid phase extraction method to quickly purify nucleic acids. This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions.  The stages of the method are: • Lyse – The cells of a sample are broken open with a lysis procedure. • Bind – A buffer solution is then added to the sample along with ethanol or isopropanol. This forms the binding solution. The binding solution is transferred to a spin column and the column is put in a centrifuge. The centrifuge forces the binding solution through a silica gel membrane that is inside the spin column. If the pH and salt concentration of the binding solution are optimal, the nucleic acid will bind to the membrane as the solution passes through.
  7. 7. • Wash – The flow-through is removed and a wash buffer is added to the column. The column is put in a centrifuge again, forcing the buffer through the membrane. This washes any remaining impurities from the membrane, leaving only the nucleic acid bound to the silica gel. • Elute – The wash buffer is removed and an elution buffer (or simply water) is added to the column. The column is put in a centrifuge again, forcing the buffer through the membrane. The elution buffer removes the nucleic acid from the membrane and it is collected from the bottom of the column.
  8. 8. Phenol–Chloroform extraction  This is a liquid-liquid extraction technique in biochemistry and molecular biology for purifying nucleic acids and eliminating proteins.  In brief, aqueous samples are mixed with equal volumes of a phenol: chloroform mixture.  After mixing, the mixture is centrifuged and two distinct phases are formed, because the Phenol: Chloroform mixture is immiscible with water.  The aqueous phase is on top because it is less dense than the organic phase (Phenol: Chloroform). The proteins will part to the lower organic phase while the nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in the upper aqueous phase.  The upper aqueous phase is pipetted off and care is taken to avoid pipetting any of the organic phase or material at the interface. This procedure is often performed multiple times to increase the purity of the DNA
  9. 9. Recent Trends in DNA Purification  Refinements of the technique include adding a chelating agent to sequester divalent cations, such as Mg2+ and Ca2+, which prevents enzymes like Dnase from degrading the DNA.  With Advancements in Robotics have resulted in scientists contemplating the use of Robots for complex processes such as DNA Purification.  We have also seen certain companies such as QIAGEN, Promega, Master Pure etc. manufacture kits for DNA Purification.  Compounds such as Guanidinium Thiocyanate or Guanidinium Hydrochloride as the chaotropic agent.
  10. 10. References • https://www.k-state.edu/hermanlab/protocols/phenolppt.html • http://www.bio-rad.com/en-us/applications- technologies/nucleic-acid-extraction-purification • www.pall.in/main/laboratory/dna-purification-methods- 52956.page • http://www.ncbi.nlm.nih.gov/pubmed/1691208 • http://lifescience.roche.com/shop/en/es/overviews/brand/nuclei c-acid-isolation-and-purification-overview • www.usfca.edu/fac-staff/dever/DNA_isolation_methods.pdf • http://www.sciencemag.org/site/products/robotfinal.xhtml