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Different methods of DNA isolation
This document discusses various DNA extraction methods, including chemical-based, physical, and enzymatic techniques, with a focus on their specific applications and efficiency. Key methods include phenol-chloroform-isoamyl alcohol (PCI), proteinase K, CTAB, silica column-based methods, and magnetic bead techniques, each having distinct characteristics based on sample type and purity. The document highlights pros and cons of each method, emphasizing the importance of selecting the appropriate technique for successful DNA isolation.
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Introduction to various DNA extraction methods and their necessity for different sample types.
Different types of DNA extraction methods are summarized, categorized into chemical and physical approaches.
Chemical methods like phenol-chloroform extraction explained, emphasizing components & procedures.
Chemical methods like phenol-chloroform extraction explained, emphasizing components & procedures.
Phenol-chloroform extraction yields high quality DNA but poses challenges such as sample amount and purity.
The rapid enzymatic method using proteinase K provides high yield but raises quality concerns.
CTAB method utilizes salts for DNA extraction with high purity but may compromise yield.
Silica gel-based DNA extraction is rapid and efficient, ensuring high-quality yields without precipitation.
Extraction using Chelex and magnetic beads is quick and minimizes contamination but may yield less DNA.
CsCl method separates DNA via centrifugation but is complex and uses hazardous substances.
Discussion on the most effective DNA extraction method based on needs and conditions.
Benefits of automation in nucleic acid extraction, highlighting efficiency and reliability.
Benefits of automation in nucleic acid extraction, highlighting efficiency and reliability.
Explains various RNA extraction methods; highlights TRIzol's effectiveness and challenges of contamination.
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Different methods of DNA isolation
- 1. Different Methods ofDNA Isolation B. Naveen Rajeshwar AAH-PB0-06
- 2. • Several differenttypes of DNA extraction methods are Phenol-chloroform isoamyl alcohol, Proteinase K, CTAB method, spin column-based methods and magnetic bead-based technique. • However, which method to use depends on the sample type and purity & yield of DNA obtained. • Depending upon the type of sample, every DNA extraction method varies. • For example, the DNA extraction method for plant DNA is different from that of the blood. • Likewise, the bacterial DNA isolation method is different from other types. • Meaning, we need varieties of DNA isolation technique for different sample Introduction
- 3. • DNA extractionmethods are broadly categorized into two categories: I. Chemical-based DNA extraction method. II. Physical DNA extraction method. • The chemical DNA extraction methods are also known as solution-based methods Different types of DNA extraction methods
- 5. Chemical or solution-basedDNA extraction method • Chemical or solution-based method uses various organic & inorganic solutions. • Main steps remain similar among all methods, viz, cell lysis, precipitation and elution. • Common chemicals used- SDS, CTAB, phenol, chloroform, isoamyl alcohol, Triton X100, guanidium thiocyanate, Tris and EDTA • The solution-based DNA extraction method is subdivided into Organic solvent-based DNA extraction- phenol and chloroform Inorganic solvent-based DNA extraction- Proteinase K • Phenol and chloroform- harmful nature- less recommended- Yet best among all • The proteinase K DNA extraction method- high DNA yield- But time-consuming. • The lower stability of the enzyme is another major issue in this method.
- 6. One of thebest methods of DNA extraction. The yield and quality of DNA obtained by the PCI method is very good if we perform it well. Also referred as a phenol-chloroform and isoamyl alcohol or PCI method of DNA extraction. Major chemicals- Lysis buffer, Phenol, chloroform, ethanol, isoamyl alcohol The lysis buffer contains Tris, EDTA, MgCl2, NaCl, SDS, and other salts. Components of lysis buffer help in the lysis of the cell membrane as well as the nuclear envelope. The organic component of the technique- phenol and chloroform denature the protein portion of cells. Phenol-chloroform method of DNA extraction
- 7. Chemical Role inDNA extraction Tris DNA is pH sensitive. Maintains pH of solution (pH=8). interacts with the LPS & permeabilizes cell membrane for easy lysis EDTA Chelating agent & blocks the activity of DNase enzyme. Dnase- lyses the DNA with help of cofactor Ca2+. Chelator EDTA blocks the cofactor binding site. Works best in combination with Tris SDS Anionic detergent. Denatures the membrane protein & break open the cell membranes and nuclear envelopes NaCl Prevents the denaturation of DNA Na+ ion of NaCl creates the ionic bond with the negative charge of DNA and neutralizes it. MgCl2 Protects DNA from mixing with other cell organelles Phenol Non polar solvent, unfolding & precipitation of protein impurities Chloroform Sharpens the fuzziness between organic & aqueous layers Isoamyl alcohol Prevent foaming between interphase. Anti-foaming agent Sodium Acetate Precipitate DNA (Na+ interacts with PO3-) Ethanol/isopropanol Helps in DNA Precipitation (Interacts with water by hydrogen bonds) TE buffer It dissolves DNA
- 8. Sample (Blood, Tissue,bacteria) Sup- Add C:I= 24:1 Add extraction buffer (Tris, EDTA, NaCl, MgCl2+, SDS, pH 8.0) Pellet-Add saturated phenol in Tris Sup-Add P:C:I= 25:24:1 Dry pellet Sup- Add ethanol & sodium acetate Pellet- Add 70% Ethanol Dissolve in TE buffer- storage Soft cell wall Mix & spin- 3000rpm DNA + debris Mix & spin- 12000rpm Helps to get distinct organic & aqueous layers Mix & spin- 12000rpm (Optional) (Washing- 2 or 3 times) Mix & spin- 12000rpm Mix & spin- 12000rpm (Precipitation) Phenol Chloroform Isoamyl alcohol DNA extraction
- 9. • Combination ofphenol, chloroform and isoamyl alcohol helps in the removal of protein. • After centrifugation, the phenol settles in the bottom of the tube with denatured protein and DNA in the aqueous phase. Some protein remains in the interphase • So care must be taken to collect nucleic acid from this method. • The collected nucleic acid is precipitated with the help of chilled alcohol (Ethanol or isoproponal). • We can add salt as well to increase the yield of the DNA (Sodium acetate) • After lysis and precipitation, we have to dissolve DNA in TE buffer in the final step. It’s an organic solution method for DNA extraction.
- 10. The PCI methodof DNA extraction is widely accepted. Forensic departments trust the PCI method rather than the Kit method. Quantity of DNA obtained- very high (800 to 900 ng with great quality) If chemicals are prepared very well and performed it sincerely, result will be good by this method. However, the amount of samples required for PCI DNA extraction is high. It is difficult to isolate DNA from the samples such as hair and nail. Also, the purity of DNA becomes a major issue if not performed well.
- 11. ֍ A combinationof a salt method as well as an enzymatic method. ֍ Here the extraction buffer is used before going further on enzymatic digestion. ֍ Here phenol, chloroform or isoamyl alcohol is not used. ֍ Instead, the enzyme proteinase K is utilized for digesting the sample. ֍ The sample is incubated with proteinase K for 2 hours at 55-60°C (digest all the protein) ֍ Immediately after enzyme digestion, sample is centrifuged & precipitated by chilled alcohol. ֍ Finally, the DNA pellet is dissolved in TE buffer. ֍ This method of DNA extraction is rapid and easy. ֍ Even the yield is very high. ֍ However, the quality of DNA is a major concern for this method. Enzymatic method of DNA extraction:
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- 13. At high saltconcentration, proteins are dehydrated, lose solubility & precipitate (removed by centrifugation) The use of salts such as sodium chloride, potassium acetate & ammonium acetate helps in DNA extraction. DNA remains in supernatant However, the method gives excellent results in combination with proteinase K. Safer method than the PCI method. One of the major limitations of the salting-out method is the purity. Though enough yield can be obtained, the quality obtained might not be good. Cell Lysis Protein Digestion Resuspension DNA precipitation DNA in supernatant Centrifugation Protein Precipitation (Proteinase K) (Salt) (Remove protein) (Ethanol) (TE buffer) Salting out method
- 14. Nowadays all theDNA extraction kits available are based on the unique chemistry of the solid/ liquid phase DNA extraction. Silica is a solid substance that binds with DNA during purification along with it. The main advantage of silica gel-based DNA extraction is that it is rapid and gives “PCR ready DNA” for downstream applications. No precipitation steps are required therefore this method is superior among all. Solid-phase DNA extraction method
- 15. Very unique anddifferent from other DNA extraction methods. PCI or proteinase K method- Many times centrifugation, collection of aqueous phase or pellets depending upon the step of extraction. The silica-based DNA extraction method works on the unique chemistry of interaction between silica and DNA. A positively charged silica particles bind with the negatively charged DNA and hold it during centrifugation. The method was first described by McCormick in 1989. However, the idea was developed in 1979, when silica was used in DNA purification by Vogelstein. Silica column-based DNA extraction method
- 16. Procedure Commercially available methodand it is most routinely used in diagnostic laboratories. Widely accepted- Good quality DNA yield & minimal simple operating system. The lysis buffer breaks the cell membrane and the nuclear envelope. The proteinase K digests all the protein. All the other impurities are removed by centrifugation. Here the DNA remains bounded with silica and other impurities pass through the silica column. Now the DNA can be washed twice for improving the purity. The impurities collected in collection tube are discarded. Finally, the DNA is dissolved into the TE buffer. The method is fast reliable, accurate and consumes less time as compared to other methods.
- 17. DNA extraction usingthe anionic resins ᴥ Other DNA extraction methods- using the anionic resins, magnetic beads & CsCl density gradient DNA extraction method. ᴥ Chelex- Product of Bio-Rad laboratories made up of styrene-divinylbenzene copolymers. ᴥ The method for using chelex first described by Walsh et al., in 1991 (Blood sample) ᴥ Chelex extraction method involves adding the Chelex resin (5%) to the sample, boiling the solution (95°C), then vortexing and centrifuging it. ᴥ The cellular materials bind to the Chelex beads, while the DNA is available in supernatant ᴥ The Chelex method is much faster and simpler than organic extraction. ᴥ It only requires one tube, which decreases the risk of DNA contamination. ᴥ Unfortunately, Chelex extraction does not yield as much quantity and the DNA yielded is single- stranded, which means it can only be used for PCR-based analyses and not for RFLP. ᴥ Chelex DNA extraction commonly used as 1st step in PCR
- 18. Another resin usedby Seligson et al., was the diethylaminoethyl. In this method, the column of the tube is filled with positive resins. The cell lysate passes through the matrix and DNA binds to the positively charged resins. By applying the low concentration salt buffer, proteins and other impurities or debris are washed off and only DNA remains into the matrix. In the final step filling the matrix with the high concentration salt buffer the DNA is eluted from the resins and precipitated using the alcohol. The method is also called DNA extraction through anion exchange chromatography. Mainly used in plasmid DNA isolation kits
- 19. Magnetic separationis based on DNA reversibly binding to a magnetic solid bead that have been coated with a DNA binding antibody, or a functional group that interacts specifically with DNA. DNA extraction buffer (Lysis) & proteinase K are needed in this technique. Lysates are the applied to magnetic beads. Positively charged magnetic beads attract the negatively charged DNA. The DNA is separated under the magnetic field. After DNA binding, beads are separated from other cellular components, washed, and the purified DNA is eluted using ethanol extraction. Rapid, simple to perform and can be automated. But more costly than other methodologies DNA extraction by magnetic beads
- 20. CsCl density gradientmethod of DNA extraction • DNA is separated based on the density of it with centrifugation. • In the high-speed centrifugation, at the isopycnic point where the density of the DNA and the gradient (CsCl) become same, the DNA band will appear. • However, the method is tedious and hard to perform as it required high speed centrifugation (1,00,000 rpm) for more than 10 hours. • Limitation of the density gradient centrifugation is the use of carcinogenic EtBr in DNA extraction. • EtBr intercalates between the strands of DNA & helps in UV visualisation • Developed by Meselson and Stahl • The CsCl is subsequently removed by dialysis or by precipitation • EtBr removed by repeated extraction with organic solvents
- 21. Which method isthe BEST?
- 22. Automated nucleic acidextraction machines have many benefits over traditional manual methods. Highly increased efficiency in DNA and RNA purification by reducing labor, time & contamination caused by human intervention. The most significant advantage is the reliability of the isolated nucleic acid. The instrument take up minimal space on the lab bench. These systems can isolate high-yield and high-purity of DNA/RNA Can process up to 32 or 96 samples per run Uses magnetic beads and strong magnets. Easy to use touch screen & user friendly interface Start & stop feature Additional options- Heating block & UV light Automated nucleic acid extraction system
- 24. 4 step process 1)Lysis and binding step 2) Washing- 3 times (last wash by alcohol) 3) Drying 4) Elution
- 25. RNA extraction ᴥ 3major techniques extensively used for RNA extraction: organic extraction, such as phenol- Guanidine Isothiocyanate (GITC)-based solutions (Trizol), silica-membrane based spin column technology, and paramagnetic particle technology. ᴥ Most commonly used method- Phenol-GITC-based organic extraction. ᴥ Yet, RNA contaminated with proteins & other cellular materials, phenol-chloroform, salts & ethanol. ᴥ Additionally, these methods require safety precautions ᴥ Silica column and paramagnetic particle based RNA isolation systems- No need of toxic organic solvents, ᴥ Relatively simple, efficient, low cost, and yield total intact RNA with low levels of contamination. ᴥ Yet have, significant levels of genomic DNA contamination. ᴥ TRIzol® reagent is an acid-guanidinium-phenol based reagent (inhibit the RNase activity ) ᴥ Ideally designed for the extraction of RNA (maintains the integrity of the RNA) ᴥ The low pH (acidic) of TRIzol® controls to separate RNA from DNA and protein.
- 26. Addition of chloroformcauses phase separation Protein- bottom organic phase DNA resolves as the interface RNA is extracted to the top aqueous phase
- 27. Reference • https://geneticeducation.co.in/phenol-chloroform-dna-extraction-basics-preparation-of-chemicals-and- protocol/ • https://geneticeducation.co.in/different-types-of-dna-extraction-methods/ •https://geneticeducation.co.in/ctab-dna-extraction-buffer-for-plant-dna-extraction/ • Dhaliwal, A. (2013). DNA extraction and purification. Mater Methods, 3, 191. • Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: a laboratory manual (No. Ed. 2). Cold spring harbor laboratory press Youtube links https://youtu.be/tCStkl9GRho https://youtu.be/tKx_L6G81NU https://youtu.be/MUM8j0vdxv8 https://youtu.be/nSTrImwajeo Thank you..!