DNA extraction

The first isolation of DNA was done in 1869 by Friedrich Miescher.[1] Currently it is a routine procedure in molecular biology or forensic analyses. For the chemical method, there are many different kits used for extraction, and selecting the correct one will save time on kit optimization and extraction procedures. PCR sensitivity detection is considered to show the variation between the commercial kits.[2]

For the various methods, see Nucleic acid methods.

History

Basic procedure
  • Cells which are to be studied need to be collected.
  • Breaking the cell membranes open to expose the DNA along with the cytoplasm within (cell lysis).
  • The solution is treated with a concentrated salt solution (saline) to make debris such as broken proteins, lipids and RNA clump together.
  • Centrifugation of the solution, which separates the clumped cellular debris from the DNA.
  • DNA purification from detergents, proteins, salts and reagents used during the cell lysis step. The most commonly used procedures are:

Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation.

After isolation, the DNA is dissolved in a slightly alkaline buffer, usually in a TE buffer, or in ultra-pure water.

Method selection

Some of the most common DNA extraction methods include organic extraction, Chelex extraction, and solid phase extraction.[3] These methods consistently yield isolated DNA, but they differ in both the quality and the quantity of DNA yielded. When selecting a DNA extraction method, there are multiple factors to consider, including cost, time, safety, and risk of contamination.

Organic extraction involves the addition of and incubation in multiple different chemical solutions;[3] including a lysis step, a phenol chloroform extraction, an ethanol precipitation, and washing steps. Organic extraction is often used in laboratories because it is cheap, and it yields large quantities of pure DNA. Though it is easy, there are many steps involved, and it takes longer than other methods. It also involves the unfavorable use of the toxic chemicals phenol and chloroform, and there is an increased risk of contamination due to transferring the DNA between multiple tubes.[4] Several protocols based on organic extraction of DNA were effectively developed decades ago,[5] though improved and more practical versions of these protocols have also been developed and published in the last years.[6]

Chelex extraction method involves adding the Chelex resin to the sample, boiling the solution, then vortexing and centrifuging it. The cellular materials bind to the Chelex beads, while the DNA is available in the supernatant.[4] The Chelex method is much faster and simpler than organic extraction, and it only requires one tube, which decreases the risk of DNA contamination. 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.[4]

Solid phase extraction such as using a spin-column based extraction method takes advantage of the fact that DNA binds to silica. The sample containing DNA is added to a column containing a silica gel or silica beads and chaotropic salts. The chaotropic salts disrupt the hydrogen bonding between strands and facilitate binding of the DNA to silica by causing the nucleic acids to become hydrophobic. This exposes the phosphate residues so they are available for adsorption.[7] The DNA binds to the silica, while the rest of the solution is washed out using ethanol to remove chaotropic salts and other unnecessary constituents.[3] The DNA can then be rehydrated with aqueous low salt solutions allowing for elution of the DNA from the beads.

This method yields high-quality, largely double-stranded DNA which can be used for both PCR and RFLP analysis. This procedure can be automated[4] and has a high throughput, although lower than the phenol-chloroform method. This is a one-step method i.e the entire procedure is completed in one tube. This lowers the risk of contamination making it very useful for forensic extraction of DNA. Multiple solid phase extraction commercial kits are manufactured and marketed by different companies; the only problem is that they are more expensive than organic extraction or Chelex extraction.

Special types

Specific techniques must be chosen for isolation of DNA from some samples. Typical samples with complicated DNA isolation are:

  • archaeological samples containing partially degraded DNA, see ancient DNA [8]
  • samples containing inhibitors of subsequent analysis procedures, most notably inhibitors of PCR, such as humic acid from soil, indigo and other fabric dyes or haemoglobin in blood
  • samples from microorganisms with thick cellular wall, for example yeast
  • samples containing mixed DNA from multiple sources

Extrachromosomal DNA is generally easy to isolate, especially plasmids may be easily isolated by cell lysis followed by precipitation of proteins, which traps chromosomal DNA in insoluble fraction and after centrifugation, plasmid DNA can be purified from soluble fraction.

A Hirt DNA Extraction is an isolation of all extrachromosomal DNA in a mammalian cell. The Hirt extraction process gets rid of the high molecular weight nuclear DNA, leaving only low molecular weight mitochondrial DNA and any viral episomes present in the cell.

Detection of DNA

A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95 °C) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to w-hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations.

Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nanometres, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has a ratio of 1.8 at 260/280 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.

DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose gel, staining with ethidium bromide (EtBr) or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.

Using the Southern blot technique, this quantified DNA can be isolated and examined further using PCR and RFLP analysis. These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which forensic scientists use for comparison, identification, and analysis.

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See also

References
  1. Dahm R (January 2008). "Discovering DNA: Friedrich Miescher and the early years of nucleic acid research". Human Genetics. 122 (6): 565–81. doi:10.1007/s00439-007-0433-0. PMID 17901982.
  2. Yoshikawa H, Dogruman-Al F, Dogruman-Ai F, Turk S, Kustimur S, Balaban N, Sultan N (October 2011). "Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples". Parasitology Research. 109 (4): 1045–50. doi:10.1007/s00436-011-2342-3. PMID 21499752.
  3. Elkins KM (2013). "DNA Extraction". Forensic DNA Biology. pp. 39–52. doi:10.1016/B978-0-12-394585-3.00004-3. ISBN 9780123945853.
  4. Butler JM (2005). Forensic DNA typing : biology, technology, and genetics of STR markers (2nd ed.). Amsterdam: Elsevier Academic Press. ISBN 9780080470610. OCLC 123448124.
  5. Marmur, J. (1961). "A procedure for the isolation of deoxyribonucleic acid from micro-organisms". Journal of Molecular Biology. 3 (2): 208–IN1. doi:10.1016/S0022-2836(61)80047-8.
  6. Salvà-Serra F, Svensson-Stadler L, Busquets A, Jaén-Luchoro D, Karlsson R, Moore ER, Gomila M (2018). "A protocol for extraction and purification of high-quality and quantity bacterial DNA applicable for genome sequencing: A modified version of the Marmur procedure". Protocol Exchange. doi:10.1038/protex.2018.084.
  7. Li, Richard (11 March 2015). Forensic biology (2nd ed.). Boca Raton. ISBN 978-1439889725. OCLC 907517669.
  8. Pääbo S (March 1989). "Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification". Proceedings of the National Academy of Sciences of the United States of America. 86 (6): 1939–43. Bibcode:1989PNAS...86.1939P. doi:10.1073/pnas.86.6.1939. PMC 286820. PMID 2928314.

Further reading
  • Sambrook, Michael R. Green, Joseph. Molecular Cloning. (4th ed. ed.). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Pr. ISBN 1936113422.
  • Forensic Biology, Richard Li, (2015) Boca Raton : CRC Press, Taylor & Francis Group. ISBN 9781439889701
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