User:SPak12/sandbox

Fossil DNA Preservation

Fossil retrieval starts with selecting an excavation site. Potential excavation sites are usually identified with the mineralogy of the location and visual detection of bones in the area. However, there are more ways to discover excavation zones using technology such as field portable x-ray fluorescence and Dense Stereo Reconstruction. Tools used include knives, brushes, and pointed trowels which assist in the removal of fossils from the earth.

To avoid contaminating the ancient DNA, specimens are handled with gloves and stored in -20°C immediately after being unearthed. Ensuring that the fossil sample is analyzed in a lab that has not been used for other DNA analysis could prevent contamination as well. Bones are milled to a powder and treated with a solution before the polymerase chain reaction (PCR) process. Samples for DNA amplification may not necessarily be fossil bones. Preserved skin, salt- preserved or air-dried, can also be used in certain situations.

DNA preservation is difficult because the bone fossilisation degrades and DNA is chemically modified, usually by bacteria and fungi in the soil. The best time to extract DNA from a fossil is when it is freshly out of the ground as it contains six times the DNA when compared to stored bones. The temperature of extraction site also affects the amount of obtainable DNA, evident by a decrease in success rate for DNA amplification if the fossil is found in warmer regions. A drastic change of a fossil's environment also affects DNA preservation. Since excavation causes an abrupt change in the fossil's environment, it may lead to physiochemical change in the DNA molecule. Moreover, DNA preservation is also affected by other factors such as the treatment of the unearthed fossil like (eg. washing, brushing and sun dring), pH, irradiation, the chemical composition of bone and soil, and hydrology. There are three perseveration diagenetic phases. The first phase is bacterial putrefaction, which is estimated to cause a 15-fold degradation of DNA. Phase 2 is when bone chemically degrades, mostly by depurination. The third diagenetic phase occurs after the fossil is excavated and stored, in which bone DNA degradation occurs most rapidly.

Methods of DNA Extraction

Once a specimen is collected from an archaeological site, DNA can be extracted through a series of processes.  One of the more common methods utilizes silica and takes advantage of polymerase chain reactions in order to collect ancient DNA from bone samples.

There are several challenges that add to the difficulty when attempting to extract ancient DNA from fossils and prepare it for analysis. DNA is continuously being split up. While the organism is alive these splits are repaired; however, once an organism has died, the DNA will begin to deteriorate without repair. This results in samples having strands of DNA measuring around 100 base pairs in length. Contamination is another significant challenge at multiple steps throughout the process. Often other DNA, such as bacterial DNA, will be present in the original sample. To avoid contamination it is necessary to take many precautions such as separate ventilation systems and workspaces for ancient DNA extraction work. The best samples to use are fresh fossils as uncareful washing can lead to mold growth. DNA coming from fossils also occasionally contains a compound that inhibits DNA replication. Coming to a consensus on which methods are best at mitigating challenges is also difficult due to the lack of repeatability caused by the uniqueness of specimens.

Silica-based DNA extraction is a method used as a purification step to extract DNA from archaeological bone artifacts and yield DNA that can be amplified using polymerase chain reaction (PCR) techniques. This process works by using silica as a means to bind DNA and separate it from other components of the fossil process that inhibit PCR amplification.  However, silica itself is also a strong PCR inhibitor, so careful measures must be taken to ensure that silica is removed from the DNA after extraction.  The general process for extracting DNA using the silica-based method is outlined by the following:

1. Bone specimen is cleaned and the outer layer is scraped off
2. Sample is collected from preferably compact section
3. Sample is ground to fine powder and added to an extraction solution to release DNA
4. Silica solution is added and centrifuged to facilitate DNA binding
5. Binding solution is removed and a buffer is added to the solution to release the DNA from the silica

One of the main advantages of silica-based DNA extraction is that it is relatively quick and efficient, requiring only a basic laboratory setup and chemicals.  It is also independent of sample size, as the process can be scaled to accommodate larger or smaller quantities.  Another benefit is that the process can be executed at room temperature.  However, this method does contain some drawbacks.  Mainly, silica-based DNA extraction can only be applied to bone and teeth samples; they cannot be used on soft tissue.  While they work well with a variety of different fossils, they may be less effective in fossils that are not fresh (e.g. treated fossils for museums).  Also, contamination poses a risk for all DNA replication in general, and this method may result in misleading results if applied to contaminated material.

Polymerase chain reaction is a process that can amplify segments of DNA and is often used on extracted ancient DNA. It has three main steps: denaturation, annealing, and extension. Denaturation splits the DNA into two single strands at high temperatures. Annealing involves attaching primer strands of DNA to the single strands that allow Taq polymerase to attach to the DNA. Extension occurs when Taq polymerase is added to the sample and matches base pairs to turn the two single strands into two complete double strands. This process is repeated many times, and is usually repeated a higher number of times when used with ancient DNA. Some issues with PCR is that it requires overlapping primer pairs for ancient DNA due to the short sequences. There can also be “jumping PCR” which causes recombination during the PCR process which can make analyzing the DNA more difficult in inhomogeneous samples.

Methods of DNA Analysis

DNA extracted from fossil remains is primarily sequenced using Massive parallel sequencing , which allows simultaneous amplification and sequencing of all DNA segments in a sample, even when it is highly fragmented and of low concentration. It involves attaching a generic sequence to every single strand that generic primers can bond to, and thus all of the DNA present is amplified.  This is generally more costly and time intensive than PCR but due to the difficulties involved in Ancient DNA amplification it is cheaper and more efficient. One method of massive parallel sequencing, developed by Margulies et al., employs bead-based emulsion PCR and pyrosequencing , and was found to be powerful in analyses of ancient DNA (aDNA) because it avoids potential loss of sample, substrate competition for templates, and error propagation in replication .

The most common way to analyze aDNA sequence is to compare it with a known sequence from other sources, and this could be done in different ways for different purposes.

The identity of the fossil remain can be uncovered by comparing its DNA sequence with those of known species using software such as BLASTN . This archaeogenetic approach is especially helpful when the morphology of the fossil is ambiguous . Apart from that, species identification can also be done by finding specific genetic markers in an aDNA sequence. For example, the American indigenous population is characterized by specific mitochondrial RFLPs and deletions defined by Wallace et al. .

aDNA comparison study can also reveal the evolutionary relationship between two species. The number of base differences between DNA of an ancient species and that of a closely-related extant species can be used to estimate the divergence time of those two species from their last common ancestor. The phylogeny of some extinct species, such as Australian marsupial wolves and American ground sloths, has been constructed by this method . Mitochondrial DNA in animals and chloroplast DNA in plants are usually used for this purpose because they have hundreds of copies per cell and thus are more easily accessible in ancient fossils.

Another method to investigate relationship between two species is through DNA hybridization. Single-stranded DNA segments of both species are allowed to form complementary pair bonding with each other. More closely related species have a more similar genetic makeup, and thus a stronger hybridization signal. Scholz et al. conducted southern blot hybridization on Neanderthal aDNA (extracted from fossil remain W-NW and Krapina). The results showed weak ancient human-Neanderthal hybridization and strong ancient human-modern human hybridization. The human-chimpanzee and neanderthal-chimpanzee hybridization are of similarly weak strength. This suggests that humans and neanderthals are not as closely related as two individuals of the same species are, but they are more related to each other than to chimpanzees .

There have also been some attempts to decipher aDNA to provide valuable phenotypic information of ancient species. This is always done by mapping aDNA sequence onto the karyotype of a well-studied closely related species, which share a lot of similar phenotypic traits. For example, Green et al. compared the aDNA sequence from Neanderthal Vi-80 fossil with modern human X and Y chromosome sequence, and they found a similarity in 2.18 and 1.62 bases per 10,000 respectively, suggesting Vi-80 sample was from a male individual . Other similar studies include finding of a mutation associated with dwarfism in Arabidopsis in ancient Nubian cotton , and investigation on the bitter taste perception locus in Neanderthals .