“Explore this students’ and researchers’ guide on the role of 70+ different chemicals used in DNA extraction and their significance in different protocols.”
Various chemicals in DNA extraction play a significant role in increasing DNA purity and yield. However, chemical composition varies from protocol to protocol. Not all the chemicals are effective for all the protocols.
DNA extraction has been an integral and crucial primarily scheme in any genetic experiment. It provides the starting material, DNA, for any experiment. Different types of DNA extraction are available for different sample types.
In addition, students need to rely on manual extraction protocols and thus they need to understand the significance of every chemical used for DNA extraction.
In this article, I will explain the function of various chemicals used in various types of DNA extraction protocols.
Stay tuned.
Key Topics:
70+ Different Chemicals used in DNA extraction
Now! What we will do is we will structure this article based on the steps and chemicals used in each step. Common DNA extraction steps are: cell lysis, DNA precipitation, washing and DNA elution.
Cell lysis:
Cell lysis is a primitive and crucial step in DNA extraction. Here, we first need to break the cell wall or membrane. The cell wall is made up of various types of components, and hence various chemical combinations have been applied to effectively lyse the cell wall or membrane.
Usually, the cell lysis buffer contains all the essential ingredients like detergents, solvents, salts and buffers. The lysis buffer and enzyme have been applied to effectively lyse the cell wall.
Buffering agents:
Buffering agents provide a constant pH environment for the enzyme to work effectively. It also protects our DNA from damage. The list of buffering agents is listed here.
| Sr No. | Buffer | Function | Protocol |
| 1 | Tris-HCl | Stabilizes pH (7.5–8.0), | CTAB, Phenol-Chloroform, SDS-based |
| 2 | TE buffer | DNA preservation, DNase inhibition | DNA storage and lysis |
| 3 | PBS | Washes cells, maintains isotonic balance | Pre-lysis washing in kits/manual methods |
| 4 | STE Buffer | Bacterial cell lysis | Bacterial DNA extraction protocols |
| 5 | EDTA buffer | DNase inhibition by chelating Mg²⁺ | CTAB, Phenol-Chloroform, SDS methods |
| 6 | SSC buffer | Stabilizes nucleic acids | Hybridization-based DNA/RNA work |
| 7 | HEPES buffer | Maintains physiological pH | Enzyme-sensitive or RNA protocols |
| 8 | Sodium acetate buffer | Aids DNA precipitation | Alcohol precipitation step |
| 9 | MES | Buffers acidic pH | Specialized RNA or acidic protocols |
| 10 | MOPS | Buffers near-neutral pH | RNA work, some eukaryotic protocols |
Detergents:
Detergents in DNA extraction also have an important role as they disrupt the cellular membrane. In our previous article, we explained the importance of detergents comprehensively. You can check out that article as well.
Detergents are categorised into anionic detergents, cationic detergents and Zwitterionic detergents, based on that they are used in various protocols. Different detergents used in DNA extraction and their function is explained here.
| Sr No. | Detergent | Function | Protocols |
| 11 | SDS | Strong anionic detergent, breaks cell membrane, denatures proteins | CTAB, SDS-based, Phenol-Chloroform |
| 12 | CTAB | Cationic detergent, removes polysaccharides | CTAB-based plant DNA extraction |
| 13 | Triton X 100 | Non-ionic detergent, gentle lysis | Animal cell, nuclei lysis, commercial kits |
| 14 | Twin 20 | Non-ionic, gentle surfactant | Washing buffers in kits, mild lysis |
| 15 | Urea | Denatures proteins and nucleoprotein complexes | Plant and fungal protocols, protein-rich samples |
| 16 | Nonidet 40 | Non-ionic detergent, disrupts lipid membranes | Mammalian cell lysis, nuclear DNA prep |
| 17 | Sarkosyl | Strong ionic detergent, solubilizes proteins | High-yield lysis protocols, (mostly for RNA prep) |
| 18 | Brij-58 | Mild non-ionic surfactant | Specialized protocols, nuclei lysis |
| 19 | CHAPS | Zwitterionic, solubilizes membrane proteins | Protein-DNA interaction studies |
| 20 | Lauryl maltoside | Mild non-ionic detergent, membrane solubilizer | Membrane-bound DNA prep |
| 21 | Triton X-114 | Phase-separating non-ionic detergent | Endotoxin removal, DNA cleanup |
| 22 | Digitonin | Selectively permeabilizes cholesterol-rich membranes | Mitochondrial DNA prep |
| 23 | Decyl maltoside | Mild detergent, maintains protein structure | DNA-protein extraction in mild lysis |
| 24 | Tergitol | Non-ionic, used for solubilizing membranes | Mild detergent in custom protocols |
Salts:
Salts have an extensive function in DNA extraction. Various types of salts are employed at different steps of DNA extraction. They are used in cell lysis– as a lysis buffer constituent, DNA precipitation, stabilizing agent and phase separator. They are also used to remove protein during extraction.
Related article: Role of Sodium Acetate in DNA Extraction.
Salts and their functions have been explained in the table below.
| Sr No | Salt | Function | Protocols |
| 25 | NaCl (Sodium chloride) | Maintains ionic strength, stabilizes and precipitates DNA | CTAB, SDS, Phenol-Chloroform |
| 26 | KCl (Potassium chloride) | Precipitates proteins, helps DNA stabilization | SDS, TE buffer protocols |
| 27 | LiCl (Lithium chloride) | Precipitates RNA, separates RNA from DNA | RNA removal in DNA extraction |
| 28 | MgCl₂ (Magnesium chloride) | Cofactor for enzymes, stabilizes DNA-DNA interactions | Enzyme-based protocols, PCR prep |
| 29 | CaCl₂ (Calcium chloride) | Helps in DNA precipitation and membrane disruption | Bacterial lysis protocols |
| 30 | Ammonium acetate | DNA precipitation, removes protein contaminants | All protocols |
| 31 | Potassium acetate | Neutralizes pH and SDS, precipitates proteins | Alkaline lysis (plasmid DNA) |
| 32 | Sodium acetate | DNA precipitation by neutralizing charges | Phenol-Chloroform, Ethanol precipitation |
| 33 | Guanidine hydrochloride (chaotropic) | Chaotropic agent, denatures proteins, aids DNA binding | Silica-column based kits (Qiagen, etc.) |
| 34 | Guanidine isothiocyanate (chaotropic) | Strong chaotrope, lyses cells, inactivates RNases | TRIzol, column-based DNA/RNA extraction |
| 35 | Cesium chloride | Density gradient centrifugation, purified plasmid DNA | Ultracentrifugation-based protocols |
| 36 | Zinc chloride (ZnCl₂) | Protein precipitation, membrane disruption | Rarely used in specialized lysis protocols |
| 37 | Barium chloride | Used occasionally for DNA precipitation | Experimental DNA purification |
| 38 | Sodium citrate | Chelating agent, maintains ionic balance | Buffer component in some extraction methods |
| 39 | Aluminum sulfate | Protein precipitation, flocculant | Rarely used |
| 40 | Sodium sulfate | Enhances DNA precipitation by removing waste | Alcohol precipitation boosters |
Reducing agents:
Reducing agents removes proteins by disrupting the disulfide bonds in proteins and also prevents oxidative damage to DNA. For example, β-Mercaptoethanol has been extensively used in plant DNA extraction.
Various reducing agents and their function is explained in the table below.
| Sr No. | Reducing agent | Function | Protocols |
| 41 | DTT (Dithiothreitol) | Reduces disulfide bonds, protects nucleic acids | Used in RNA/DNA extractions to prevent degradation |
| 42 | β-Mercaptoethanol | Reduces disulfide bonds, protects nucleic acids | Plant DNA extraction |
| 43 | Sodium bisulfite | Converts unmethylated cytosines to uracil | DNA methylation analysis |
| 44 | Polyvinylpyrrolidone | Binds polyphenols, protects DNA | Plant DNA extraction |
| 45 | PVPP (Polyvinylpolypyrrolidone) | Binds polyphenols, prevents DNA degradation | Plant DNA extraction |
| 46 | Bovine Serum Albumin | Stabilizes DNA during extraction and processing | Any DNA extraction, not mandatory |
Enzymes:
An enzyme is applied to improve the quality of DNA. It denatures the protein part, such as histone and other proteins and removes it from the DNA. Proteinase K is a common enzyme used in various DNA extraction protocols.
You can read our previous article to learn more about proteinase K-based DNA extraction. All the enzymes and their applications are explained in the table below.
| Sr No | Enzyme | Function | Protocols |
| 47 | Proteinase K | Degrades proteins, removes protein contamination | Used in all DNA extraction protocols |
| 48 | RNase A | Degrades RNA, removes RNA contamination | DNA extractions requiring RNA removal (e.g., plasmid DNA isolation) |
| 49 | RNase H | Degrades RNA strand in RNA-DNA hybrids | Used in RNA/DNA hybridization studies, cDNA synthesis |
| 50 | Lysozyme | Breaks down bacterial cell walls | Common in bacterial DNA extractions |
| 51 | Cellulase | Breaks down cellulose in plant cells | Used in plant DNA extractions, especially for tough plant tissues |
| 52 | Lecticase | Degrades lectins, carbohydrate-binding proteins | Used in plant tissue DNA extractions (rarely used) |
| 53 | Zymolyase | Breaks down yeast cell walls | Used in yeast DNA extractions |
| 54 | Pepsin | Degrades proteins, particularly collagen | Used in animal tissue DNA extractions (e.g., biopsies) |
| 55 | Trypsin | Cleaves proteins into peptides, aids in cell lysis | Used in animal tissue extractions, often in cell cultures |
| 56 | Pronase | Degrades proteins into peptides and amino acids | Used in general DNA extraction protocols, especially for degraded samples |
| 57 | Chitinase | Breaks down chitin in fungal and insect cell walls | Used in fungal DNA extractions (e.g., yeast or mold) |
Organic solvents for phase separation:
Organic solvents like phenol, chloroform and isoamyl alcohol denature proteins, separate two phases to isolate DNA from the aqueous phase, effectively.
Common organic solvents and their function is explained here.
| Sr No | Organic solvent | Function | Protocols |
| 58 | Phenol | Denatures proteins, separates organic and aqueous phases | PCI |
| 59 | chloroform | Further separates organic and aqueous phases, removes lipids | PCI |
| 60 | Isoamyl alcohol | Reduces foam formation during phenol-chloroform extraction | PCI |
| 61 | Trizol | Solubilizes cells and tissues, separates RNA, DNA, and proteins | Used in RNA/DNA extraction, especially in mixed tissue samples |
| 62 | Butanol | Removes lipids, used for phase separation | Rarely used |
| 63 | Ethyl acetate | A solvent for phase separation, removes contaminants | Clean up samples |
Precipitating agents and washing:
Precipitating agents precipitate DNA into a visible, thread-like structure. Two common precipitation agents are alcohol and salts. I have explained the precipitation process, in detail in our previous article.
Alcohol is also used for washing DNA precipitates. This improves the quality of DNA. You can also read our previous article to know more.
Related articles:
You can read it to strengthen your knowledge. Various precipitating agents and their function is explained here.
| Sr No | Precipitating agent | Function | Protocols |
| 64 | Ethanol | Precipitates and purifies DNA | Used in alcohol-based DNA precipitation |
| 65 | Isopropanol | Precipitates DNA | Common in DNA precipitation |
| 66 | Methanol | Precipitates nucleic acids | Occasionally used in RNA/DNA extractions |
| 67 | 2-propanol | Precipitates DNA | Used in DNA precipitation |
| 68 | Polyethylene glycol | Precipitates DNA/RNA, removes contaminants | Used in yeast plasmid isolation |
| 69 | Glycogen | Carrier for DNA precipitation | Added for small DNA yields |
| 70 | NaCl (Sodium chloride) | Stabilizes nucleic acids | Facilitates DNA precipitation |
| 71 | LiCl (Lithium chloride) | Precipitates RNA, RNA/DNA separation | Used in RNA isolation, sometimes in DNA extractions |
| 72 | Ammonium acetate | Aids DNA precipitation | Used in alcohol-based DNA precipitation |
| 73 | Potassium acetate | Neutralizes nucleic acids, aids precipitation | All DNA extraction |
| 74 | Sodium acetate | Neutralizes and precipitates nucleic acids | All DNA extraction |
Water:
Last but not the least, water— nuclease free water has been extensively used not only during DNA extraction but during all the experimental steps. In DNA extraction, it is used to prepare buffers, solutions and even precipitate the DNA.
Wrapping up:
We explained the function of 75 different chemicals!
If you are new or a beginner in the field of DNA extraction, this complete guide on 70+ different chemicals used in DNA extraction will definitely improve your extraction knowledge and help you to make your DNA extraction experiments better.
However, choosing the right DNA extraction protocol, chemical combination or even chemicals needs experience and expertise in this field.
I hope you like this article. Do share it and subscribe to Genetic Education.
Sources:
Dairawan, M. and Shetty, P.J. (2020). The Evolution of DNA Extraction Methods. [online] American Journal of Biomedical Science & Research. Available at: https://biomedgrid.com/fulltext/volume8/the-evolution-of-dna-extraction-methods.001234.php.
