Extraction of DNA is a laboratory technique used to obtain the deoxyribonucleic acid (DNA) from biological samples, such as archaeological and historical skeletal material (related to skeleton/bones), mummified tissues, old collections of non-frozen medical specimens, preserved plant remains, ice and permafrost cores (permanently frozen earth), etc. Archaeology refers to the scientific study of material remains of past human life and activities. A mummified tissue or body is a well-preserved dead body/tissue of a human being or an animal by unusual natural conditions or embalming, a preservation process practiced by the ancient Egyptians.
Genes, or segments of DNA, are considered the building blocks of life because they provide instructions for all the cells in the body. DNA is a long thread-like molecule made up of large numbers of nucleotides. Nucleotides are molecules composed of a nitrogen base, a 5-carbon sugar, and one or more phosphate groups. The sequence of bases in DNA serves as the carrier of genetic (hereditary) information. Long strands of nucleotides form nucleic acids.
Uses: Extraction of nucleic acids from well-preserved tissues or mummified tissues is considered important, especially in the field of anthropology, the scientific study of the origin, behavior, and the physical, social, and cultural development of humans in relation to time and space. It is also of importance in the study of ancient life forms existing in prehistoric or geologic times, as represented by the fossils of plants, animals, and other organisms (known as paleontology). To study the emergence of disease-causing organisms, such as bacteria, viruses, and fungi (pathogens), and their progress since ancient times, also requires nucleic acids. DNA extraction from ancient tissues has found its use even in science or technology of the investigation and establishment of facts or evidence in a court of law (known as forensic science).
Sample: DNA of mummified tissues may be extracted from several types of biological samples, such as bones, teeth, muscles, skin, hair roots, the brain, blood vessels, genitals, lungs, etc. However, extracting nucleic acids from mummified tissues is significantly difficult, as the amount of DNA retained in the mummy is assumed to be minute, degraded, chemically modified, and contaminated due to various factors (e.g. environment) over a long period of time.
Extraction or removal of ancient DNA (aDNA) from mummified tissues or the sample of interest is performed with extreme caution to avoid contamination from other DNA sources. It is performed very gently, as the aDNA is assumed to be minute, degraded and chemically modified. Thus, several modifications are applied to the basic method, so as to achieve efficient extraction. Isolation of aDNA is further needed for genetic analysis, which is used for historical, scientific, medical, or forensic purposes. The basic outline of the aDNA extraction process involves collection of the tissue, cell lysis, protein precipitation, and DNA precipitation, which is described below.
Collection of the tissue (excision): The surface of the site where the tissue is going to be collected is cleaned and washed with pure water that is free from impurities and organisms (distilled water). The tissues are obtained from deep sites and cut into small pieces. The sample size is determined based on the degree of degradation (damage) and the amount of DNA retained in the mummified tissue.
Break open (lyse) the cells of the tissue: The small pieces of tissue are finely cut (minced), grinded, or sonicated to gently break open the cells. Sonication is the process of applying sound waves (ultrasound) to break down a material. The powdered or ground tissue is suspended uniformly without any clumps in a digestion buffer, a solution of chemical compounds that resists changes in pH (acidity or alkalinity) and breaks down the cell walls.
Protein removal (deproteinization): Several chemical and enzymatic methods are available to remove the contaminating proteins, ribonucleic acid (RNA), and other macromolecules. The lysed cells are mixed with enzymes, such as proteinase K or guanidine thiocyanate (GTC). These enzymes digest the proteins and remove the contaminations from the nucleic acid preparations. In addition, they rapidly inactivate nucleases, which are enzymes that would degrade the DNA if left during the extraction process. Along with the addition of enzymes, a chemical substance, sodium dodecyl sulfate (SDS), is also added to denature (changes the natural quality of a substance) the proteins, and to aid the proteinase K to digest the proteins.
Digested cellular proteins are removed during precipitation, a process in which the proteinaceous material gets clumped together and easily removed. This can be achieved by using either organic solvents (phenol/chloroform) or nonorganic solvents. A solvent is a solution capable of dissolving other substances. Organic compounds are carbon-containing chemicals. The denatured/digested protein solution is mixed vigorously with the phenol/chloroform mixture or ammonium acetate (an organic solvent) by vortexing (spiral motion of fluid). This is followed by centrifugation, a process of spinning at high speed that separates the solids and liquids within the solution mixture by gravity. The upper layer of the solution mixture, which contains the aDNA, is transferred to another tube. The precipitant containing the precipitated proteins is then discarded.
Precipitation of DNA: The DNA (aDNA) solution is mixed with cold isopropanol (an alcohol). As the aDNA is not soluble in the alcohol, the aDNA clumps together and can be seen as long, white stringy fibers. The precipitated aDNA is then washed with ethanol to remove salts and/or solvents previously added, as well as small organic molecules. Next, the clean aDNA is air-dried and suspended in a buffer solution, which can be stored indefinitely at -20(C for use in various genetic analysis techniques.
DNA detection and quantitation: The presence of isolated and purified aDNA is confirmed with the electrophoresis method. Electrophoresis is a technique in which charged particles migrate under the influence of an applied electric field in a gelatinous medium made of tiny particles dispersed in a continuous phase. Usually ethidium bromide or a fluorescent dye is used as a marker, as it reacts with the DNA and can be seen under ultraviolet (UV) light. Thus, the amount of DNA isolated can be estimated by the intensity of the fluorescence emitted by the DNA markers.
Alternatively, the DNA may be quantitated (precisely measured). For example, the amount of aDNA can be measured by spectrophotometric analysis, a technique that measures the light energy transmitted or reflected by aDNA.
Polymerase chain reaction (PCR): The clean and nondegraded DNA extracted from the mummified tissues is usually very minimal and may be insufficient for further use in several genetic analysis techniques. This may be overcome by using PCR, an efficient and sensitive laboratory technique that amplifies (by replication) a specific sequence of DNA into billions of its copies. The amplification process is done in the presence of sequence specific oligonucleotide primers and DNA polymerase enzyme at a controlled temperature. An oligonucleotide primer is a sequence of nucleotides, usually of 20-50 bases, that is complementary to a target DNA sequence. Oligonucelotides serve as a starting point for DNA replication. Nucleotides are molecules composed of a nitrogen base, a 5-carbon sugar, and one or more phosphate groups. DNA polymerase is an enzyme that creates new DNA strands using preexisting DNA strands as a template, thereby assisting in DNA replication. A fluorescent marker is also incorporated into the PCR product during the amplification process. This assists in visualizing the PCR products at a later stage.
Advantages: Suitable aDNA may be extracted from both bones and soft tissues. However, skeletal remains are preferable when source material is severely degraded. According to aDNA extraction and analysis studies, the phenol/chloroform-SDS-isopropanol method is the preferred method to extract DNA from tissues preserved for thousands of years.
Extinction: Some of the ongoing research includes the impacts of climate change, human evolution, and the evolutionary factors in relation to the extinction of species. These, species include Ice Age brown bears, bison, horses, cave lions, and New Zealand moa, as well as ancient humans. Evolution is the process of change in the inherited characteristics of an organism from one generation to another.
Neanderthals: Neanderthals are specimens of the Homo genus, the same as humans, who inhabited Europe and Central Asia as early as 350,000-500,000 years ago. It was debated for many years whether Neanderthals are a predecessor to modern humans. DNA was extracted from the remains of Neanderthals and analyzed. The results indicate that Neanderthals may have shared a common ancestor in the Homo genus several hundred thousand years ago; however, it was proved that they were not predecessors of modern humans.
Evolutionary process: Evolution is the process of change in the inherited characteristics of an organism from one generation to another. Ancient DNA (aDNA) may be used to study how populations and species have changed over time, as well as the response to the impact from various major environmental events, such as mass extinctions, climate change (e.g. global warming), and human impact. The study of evolutionary process also includes paleoecology and phylogenography. Paleoecology is the branch of ecology that deals with the interaction between ancient organisms and their environments. Phylogenography refers to a method of interpreting the observed distribution of phenotypic (observable characteristics) and genetic differences in certain species of organisms from various historical recordings. This allows researchers to study the process that influences genetic variations in a particular region
Population genetics: Population genetics is the study of the genetic composition of populations in order to understand the possible evolutionary forces that select for a particular gene. The aDNA, obtained from the preserved genetic records of human, animal, plant, and sedimentary material, can be utilized to study and understand evolutionary forces on population groups. Some of the evolutionary forces affecting the population genetics are natural selection, gene flow, and genetic drift. Natural selection refers to survival of the organisms that are best adapted to their environments and will live to reproduce the most viable offspring. Gene flow is the transfer of genetic material between separate populations. Genetic drift is the random change in the genetic composition of a population due to chance events.
The aDNA sequence variation may be used to construct a phylogenetic or evolutionary tree; such as a diagram and/or tree showing the evolutionary relationship between individual sequences. Therefore, aDNA serves as a phylogenetic tool to analyze the events in human prehistory. Moreover, the geographical distribution of the lineages on a tree may be used to detect prehistoric movements from one region to another, thereby facilitating the study of the migration of humans over time.
Pathogenetic detection: The emergence of disease-causing organisms, such as bacteria, viruses, and fungi (pathogens), and their progress since ancient times, may be studied using the aDNA from the organisms, as well as from humans. For example genetic material was extracted from the tooth pulp (soft portion of the inner part of tooth) of unerupted teeth from the remains of a buried human child. This material was identified as genetic material of Yersinia pestis, bacterium that causes The Plague. The Plague is a highly infectious, fatal disease that caused millions of deaths. Thus, confirmation of ancient plague was achieved from historically identified victims, thereby confirming the presence of the disease.
Forensics: The aDNA analysis helps for confirmatory identification of human remains, which may assist in criminal cases. It may also be used to provide evidence about the identity of crime victims. Additionally, the aDNA analysis may aid in the identification of the remains of victims of mass disaster.
Determination of sex: The aDNA extracted from the skeletal remains may be analyzed to determine an organism's sex. This is especially useful for juvenile and fragmentary remains when it is difficult, or impossible, to establish the type of sex from morphological features.
Detection of gene deficiency: Analysis of aDNA has also helped in identifying the origins and diversity of variations in DNA (mutations) that causes certain genetic disorders. For example, red blood cell genetic disorders, such as sickle cell anemia and thalassemia, were found from the remains of Egyptian mummies and also from the skeletal remains in Turkey. The types of mutations in these disorders aided in understanding their origins and diversity across populations.
Deoxyribonucleic acid (DNA) from ancient remains may be unable to undergo polymerase chain reaction (PCR) amplification as a result of various factors, such as degradation of the DNA, contamination, and chemical modifications, which may vary among the burial sites. Some of the chemical modifiers include humic acid, fulvic acid, hidroxyapatite, and tannin. Humic acid and fulvic acid are complex chemicals derived from humus, a major component of residual decaying organic matter in the soil. Hidroxyapatite is the main mineral component of bone. Tannins are chemical substances found in plants, which can transform certain proteins of animal tissue into compounds that resist decomposition.
Further studies are required to analyze and understand how the ancient preserved DNA is damaged over time, as well as the likely impact on the accuracy of recovered genetic information. The DNA isolated from different tissues, such as hair, bone, teeth etc., needs to be studied to observe how burial and preservation under a variety of conditions has altered them.
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