DNA is the short form for deoxyribonucleic acid, which forms the genetic basis of all living organisms. It is present in each and every cell of the organism (with obvious exceptions) and determines all the cellular functions. The ability to isolate DNA from the cells and tissues is key to the study of genetic diseases, development of diagnostic tests, drug development, forensic studies, sequencing techniques, determination of paternity, etc. The first DNA isolation was done by Friedrich Miescher in the year 1869. Density gradient centrifugation-based methods for DNA isolation was developed in 1958 by Meselson and Stahl. Since then, there have been gradual improvements in the methods used for DNA isolation with considerable development in the last decade. Today, DNA extraction procedure forms the basis and is a routine procedure in most molecular biology techniques.
Conventional DNA isolation methods:
The conventional methods of DNA isolation often follow the same basic steps. The first step is often the physical or mechanical lysis of the cells followed by removal of the membrane lipids. This is followed by the denaturation and removal of cellular proteins. The remaining cellular contaminants and the RNA are removed. In the final step, the DNA is eluted in high alkali or double-distilled water. The most commonly used DNA isolation methods include extraction using organic solvents, solid-phase extraction and chelex extraction. These methods are not consistent and often yield DNA that differs in quality and quantity.
Criteria for selection of DNA isolation methods:
It is essential to choose the correct method of DNA isolation to save the time of optimization and execution of the experiment. The factors to be considered include:
- Sample type and origin
- Sample preparation method employed
- Downstream application of the extracted DNA
- Humic acid content in the sample
- Quantity of the sample
- Expected yield of DNA
The employed method for DNA isolation should efficiently extract the DNA from the sample in adequate quality and quantity coupled to successful removal of contaminants and other hindering substances.
Nanotechnology methods for DNA isolation:
Classical and conventional methods of DNA isolation are often time-consuming, uses toxic chemicals, ethanol and organic solvents and has multiple steps. For the separation and purification of biomolecules, the employment of nanoparticles technology is very promising, especially the use of magnetic nanoparticles. The use of nanoparticles in enhancing the quality and quantity of extracted DNA has been attributed to their enhanced ability to lyse the membrane, inhibit the restriction enzymes and in precipitating the proteins. Such techniques do not require the need of centrifugation procedures, are cheap, time-saving and do not require the use of toxic chemicals. Magnetic nanoparticles are made of a magnetic material like iron, nickel and cobalt and a chemical constituent that has functionality. The technique is dependent on the ability of uncoated or naked nanoparticles to bind to the DNA in a reversible fashion under certain circumstances. The bound DNA can then be extracted using conditions of elution. Several research articles have reported nanoparticle-based DNA isolation methods from bacterial cells, soil, saliva, semen, plants, blood, etc.
1. DNA isolation from soil
- Sieved soil is mixed with lysis buffer and incubated in rotating conditions.
- The mixed sample is centrifuged and the supernatant is mixed with silica magnetite nanoparticles and the binding buffer.
- The DNA binds to the magnetic nanoparticles.
- The nanoparticles with the bound DNA are immobilized using a magnet and are washed with ethanol and left to air dry.
- DNA can then be eluted from the particles using elution buffer twice and can be used for downstream applications.
- The method is cheap, rapid and provides DNA of superior quantity and similar quality as isolated using conventional methods.
2. DNA isolation from bacteria
- The magnetic nanoparticles of 200nm diameter were employed for the isolation of DNA from bacterial cells.
- The procedure follows the routine steps where the bacterial cells are lysed to release the nucleic acid.
- Gram-positive bacteria will have an extra step of cell wall digestion which is otherwise not required with Gram-negative bacteria.
- In addition of the magnetic nanoparticles, the DNA will bind to the particles.
- The nanoparticles are then washed with ethanol to remove the contaminants.
- The DNA is then eluted from the nanoparticles and are used for further analysis.
- When compared to conventional DNA isolation protocols, the methods employing nanoparticles exhibited better intractability.
3. DNA isolation from dried semen and saliva samples
- Saliva and semen are valuable samples for forensic studies to identify the criminals.
- As the samples often get dried in the crime scenes, the procedures for isolating DNA from dried semen and saliva samples are often very time consuming and costly so that it can safely contribute in the preliminary stage of investigation.
- Nanoparticle based methods have been devised to isolate DNA from such samples.
- Swabs of semen and saliva were allowed to dry and treated with TE buffer and acidulated water to extract the spermatozoa and epithelial cells respectively.
- The mixture will be centrifuged to settle down the cells as a pellet.
- The supernatant will be discarded and to the pellet, cell lysis buffer will be added.
- To the incubated cells, magnetic nanoparticles will be added followed by binding buffer.
- The mixture will be incubated for the magnetic-biomolecule conjugate to form.
- With the use of external magnetic field, the nanoparticles with the bound biomolecules can be separated and washed with ethanol.
- The bound nucleic acid can be eluted using elution buffer and the extracted DNA can be used for further analysis.
The magnetic-nanoparticle based methods for DNA isolation is highly reliant on cell lysis, adsorption of DNA to the beads, separation of the nanoparticle-biomolecule complex using magnetic field and washing of the complexes to remove the inhibitors and contaminants which can hinder DNA isolation. The magnetic nanoparticles reversibly bind the nucleic acids through the combined action of electrostatic force, dehydration and hydrogen bond formation. This technique has been used to successfully isolate DNA from plant samples, blood, bacteria, forensic tissue, etc. The technique is feasible, precise, convenient, rapid, noninvasive, and painless.
- Al-Jeboory MR, Al-Jailawi MH, Al-Obaedi AM, Al-Jeboory SR, 2015. Improvement of DNA extraction methods by ZnO and TiO2 nanoparticles. International Conference on Medical Genetics, Cellular and Molecular Biology, Pharmaceutical and Food Sciences.
- Sebastianelli A, Sen T, Bruce IJ, 2007. Extraction of DNA from soil using nanoparticles by magnetic bio separation. Letters in Applied Microbiology. 46: 488-491.
- Chen Y, Lin J, Jiang Q, Chen Q, Zhang S, Li L, 2016. A magnetic nanoparticle-based nucleic acid isolation and purification instrument for DNA extraction of Escherichia coli O157:H7. Journal of Nanoscience and Nanotechnology. 16: 2296-2300.
- Bagban MA, Mansuri R, Jain N, 2018. A novel nanoparticle-based method to isolate DNA from dried saliva and semen samples. Journal of Advanced Medical Sciences and Applied Technologies. 4(1): 13-20.
- Yi L, Huang Y, Wu T, Wu J, 2013. A magnetic nanoparticle-based method for DNA extraction from the saliva of stroke patients. Neural Regeneration Research. 8(32): 3036-3046.