Isolation and Purification of DNA from Complicated Biological Samples
The isolation of nucleic acids from a biological sample is an important step for many molecular biology applications and medical diagnostic assays. This chapter describes an efficient protocol using established acidic CTAB (with a pH value of 5.0 to 6.8)
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Introduction Nucleic acid sequences have a variety of applications in the field of molecular biology. They are a valuable tool in many analytical and application techniques used in the field of molecular biology, health, medicine (gene therapy, diagnostics, and recombinant protein expression), forensics, and food science. Some examples of these techniques include next-generation sequencing applications, genotyping with DNA fingerprinting, detection of pathogens, and forensic identification of biological samples and environmental samples contaminated with different biological entities [1–8]. To be used as a diagnostic tool, the target nucleic acid sequence should be free of contaminants that inhibit PCR and other downstream applications. Such contaminants chemically or mechanically block or inhibit chemical and enzymatic reactions, including denaturation and hybridization of nucleic acids, and other applications used in molecular biology methods. Contaminants can also degrade or modify the nucleic acid. These include high-molecular substances, such as polysaccharides and polyphenols, as well as substances of lower molecular weight, such as pigments, secondary
Pascale Besse (ed.), Molecular Plant Taxonomy: Methods and Protocols, Methods in Molecular Biology, vol. 2222, https://doi.org/10.1007/978-1-0716-0997-2_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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metabolites, lipids, humic substances, low-molecular enzyme inhibitors, and oligonucleotides. Therefore, in order to be able to use nucleic acids from biological materials for further analysis, it is important that these substances are eliminated entirely from the sample. Isolating DNA or RNA that is sufficiently purified from contaminants is complicated by the diversity and complex composition of biological material from which DNA and RNA are isolated. Biological material consists of cells and tissues. Cells in liquid media, such as blood, lymph, milk, urine, and feces, and cells in culture, on an agarose or polyacrylamide gel, in soil, or in solution, usually include significant amounts of contaminants that must be removed from the DNA or RNA before molecular biology experiments. The presence of chemical or mechanical crosslinks between DNA chains and with contaminants interweaving with DNA leads to partial or complete inhibition of DNA denaturation and the appearance of artifacts. The quality of nucleic acids directly influences problems and artifacts produced by molecular biology procedures downstream. Thus, for efficient DNA amplification, for example, using the PCR method or isothermal DNA amplification, complete separation of nucleic acid strands at all lengths is required. A variety of DNA extraction and purification methods have been developed [9–23], and are known for different characteristics. Ionic ion exchange resins were used to purify a nucleic acid already in 1953 [24]. Nucleic acids, proteins, and other contaminants are bound on a solid support by anion exchange. Nucleic acids are then el
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