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=== Fossil DNA Preservation ===

[[Fossil]] retrieval starts with selecting an [[Excavation (archaeology)|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]]<ref>{{Cite journal|last=Cohen|first=David R.|last2=Cohen|first2=Emma J.|last3=Graham|first3=Ian T.|last4=Soares|first4=Georgia G.|last5=Hand|first5=Suzanne J.|last6=Archer|first6=Michael|date=October 2017|title=Geochemical exploration for vertebrate fossils using field portable XRF|url=http://www.sciencedirect.com/science/article/pii/S0375674216304447|journal=Journal of Geochemical Exploration|volume=181|pages=1–9|doi=10.1016/j.gexplo.2017.06.012}}</ref> and Dense Stereo Reconstruction<ref>{{Cite journal|last=Callieri|first=Marco|last2=Dell'Unto|first2=Nicolo|last3=Dellepiane|first3=Matteo|last4=Scopigno|first4=Roberto|last5=Söderberg|first5=Bengt|last6=Larsson|first6=Lars|date=2011|title=Documentation and Interpretation of an Archeological Excavation: an experience with Dense Stereo Reconstruction tools|url=http://lup.lub.lu.se/record/2062881|journal=[Host publication title missing]|publisher=Eurographics Association|pages=33–40|isbn=9783905674347}}</ref>. Tools used include [[Knife|knives]], [[Brush|brushes]], and pointed [[Trowel|trowels]] which assist in the removal of fossils from the earth<ref name=":9">{{Cite book|title=Digging Up Bones: The Excavation, Treatment, and Study of Human Skeletal Remains|last=Brothwell|first=Don R.|publisher=Cornell University Press|year=1981|isbn=9780801498756|location=|pages=2 - 3}}</ref>.

To avoid [[Contamination|contaminating]] the [[ancient DNA]], [[Specimen|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<ref name=":9" /><ref name=":10" />. Bones are [[milled]] to a powder and treated with a solution before the polymerase chain reaction (PCR) process<ref name=":10">{{Cite journal|last=Scholz|first=Michael|last2=Bachmann|first2=Lutz|last3=Nicholson|first3=Graeme J.|last4=Bachmann|first4=Jutta|last5=Giddings|first5=Ian|last6=Rüschoff-Thale|first6=Barbara|last7=Czarnetzki|first7=Alfred|last8=Pusch|first8=Carsten M.|date=2000-06-01|title=Genomic Differentiation of Neanderthals and Anatomically Modern Man Allows a Fossil–DNA-Based Classification of Morphologically Indistinguishable Hominid Bones|url=http://www.sciencedirect.com/science/article/pii/S0002929707635447|journal=The American Journal of Human Genetics|volume=66|issue=6|pages=1927–1932|doi=10.1086/302949}}</ref>. Samples for [[DNA amplification]] may not necessarily be fossil bones. Preserved skin, salt- preserved or air-dried, can also be used in certain situations<ref>{{Cite journal|last=Yang|first=H.|last2=Golenberg|first2=E. M.|last3=Shoshani|first3=J.|date=June 1997|title=Proboscidean DNA from museum and fossil specimens: an assessment of ancient DNA extraction and amplification techniques|url=https://www.ncbi.nlm.nih.gov/pubmed/9332711|journal=Biochemical Genetics|volume=35|issue=5-6|pages=165–179|issn=0006-2928|pmid=9332711}}</ref>.

DNA preservation is difficult because the bone [[fossilisation]] degrades and DNA is chemically modified, usually by [[bacteria]] and [[Fungus|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<ref>{{Cite journal|last=Scholz|first=Michael|last2=Bachmann|first2=Lutz|last3=Nicholson|first3=Graeme J.|last4=Bachmann|first4=Jutta|last5=Giddings|first5=Ian|last6=Rüschoff-Thale|first6=Barbara|last7=Czarnetzki|first7=Alfred|last8=Pusch|first8=Carsten M.|date=2000-06-01|title=Genomic Differentiation of Neanderthals and Anatomically Modern Man Allows a Fossil–DNA-Based Classification of Morphologically Indistinguishable Hominid Bones|url=http://www.sciencedirect.com/science/article/pii/S0002929707635447|journal=The American Journal of Human Genetics|volume=66|issue=6|pages=1927–1932|doi=10.1086/302949}}</ref>.

=== Methods of DNA Extraction ===
=== Methods of DNA Extraction ===
Once a specimen is collected from an archaeological site, DNA can be extracted through a series of processes[11].  One of the more common methods utilizes silica and takes advantage of [[polymerase chain reaction|polymerase chain reactions]] in order to collect [[ancient DNA]] from bone samples[3].
Once a specimen is collected from an archaeological site, DNA can be extracted through a series of processes.<ref name=":4">{{Cite journal|last=Hagelberg|first=Erika|last2=Clegg|first2=J. B.|date=1991-04-22|title=Isolation and Characterization of DNA from Archaeological Bone|url=http://rspb.royalsocietypublishing.org/content/244/1309/45|journal=Proceedings of the Royal Society of London B: Biological Sciences|language=en|volume=244|issue=1309|pages=45–50|doi=10.1098/rspb.1991.0049|issn=0962-8452|pmid=1677195}}</ref>  One of the more common methods utilizes silica and takes advantage of [[polymerase chain reaction|polymerase chain reactions]] in order to collect [[ancient DNA]] from bone samples.<ref name=":5">{{Cite journal|last=Rohland|first=Nadin|last2=Hofreiter|first2=Michael|date=July 2007|title=Ancient DNA extraction from bones and teeth|url=http://www.nature.com/nprot/journal/v2/n7/full/nprot.2007.247.html?foxtrotcallback=true|journal=Nature Protocols|language=en|volume=2|issue=7|pages=1756–1762|doi=10.1038/nprot.2007.247|issn=1754-2189}}</ref>


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 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[8]. The best samples to use are fresh fossils as uncareful washing can lead to mold growth[11]. DNA coming from fossils also occasionally contain a compound that inhibits DNA replication[6]. 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[8].
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 pair|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.<ref name=":6">{{Cite journal|last=Handt|first=O.|last2=Höss|first2=M.|last3=Krings|first3=M.|last4=Pääbo|first4=S.|date=1994-06-01|title=Ancient DNA: Methodological challenges|url=https://link.springer.com/article/10.1007/BF01921720|journal=Experientia|language=en|volume=50|issue=6|pages=524–529|doi=10.1007/BF01921720|issn=0014-4754}}</ref> The best samples to use are fresh fossils as uncareful washing can lead to [[mold]] growth.<ref name=":4" /> DNA coming from fossils also occasionally contains a compound that inhibits DNA replication.<ref name=":7">{{Cite journal|last=Höss|first=M|last2=Pääbo|first2=S|date=1993-08-11|title=DNA extraction from Pleistocene bones by a silica-based purification method.|url=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC309938/|journal=Nucleic Acids Research|volume=21|issue=16|pages=3913–3914|issn=0305-1048|pmid=8396242}}</ref> 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.<ref name=":6" />


[[DNA separation by silica adsorption|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|polymerase chain reaction (PCR)]] techniques.[6]  This process works by using silica as a means to bind DNA and separate it from other components of the fossil process that inhibit [[polymerase chain reaction|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.[12]  The general process for extracting DNA using the silica-based method is outlined by the following:
[[DNA separation by silica adsorption|Silica-based DNA extraction]] is a method used as a purification step to extract DNA from archaeological bone [[Artifact (archaeology)|artifacts]] and yield DNA that can be amplified using [[polymerase chain reaction|polymerase chain reaction (PCR)]] techniques.<ref name=":7" /> This process works by using silica as a means to bind DNA and separate it from other components of the fossil process that inhibit [[polymerase chain reaction|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.<ref>{{Cite journal|last=Yang|first=Dongya Y.|last2=Eng|first2=Barry|last3=Waye|first3=John S.|last4=Dudar|first4=J. Christopher|last5=Saunders|first5=Shelley R.|date=1998-04-01|title=Improved DNA extraction from ancient bones using silica-based spin columns|url=http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-8644(199804)105:43.0.CO;2-1/abstract|journal=American Journal of Physical Anthropology|language=en|volume=105|issue=4|pages=539–543|doi=10.1002/(SICI)1096-8644(199804)105:43.0.CO;2-1|issn=1096-8644}}</ref>  The general process for extracting DNA using the silica-based method is outlined by the following:<ref name=":5" />
# Bone specimen is cleaned and the outer layer is scraped off
# Bone specimen is cleaned and the outer layer is scraped off
# Sample is collected from preferably compact section
# Sample is collected from preferably compact section
Line 10: Line 18:
# Silica solution is added and centrifuged to facilitate DNA binding
# Silica solution is added and centrifuged to facilitate DNA binding
# Binding solution is removed and a buffer is added to the solution to release the DNA from the silica
# 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 [[DNA separation by silica adsorption|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.[3]  However, this method does contain some drawbacks.  Mainly, [[DNA separation by silica adsorption|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.[3]
One of the main advantages of [[DNA separation by silica adsorption|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, [[DNA separation by silica adsorption|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 [[Museum|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.<ref name=":5" />

⚫
[[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.<ref name=":4" /> This process is repeated many times, and is usually repeated a higher number of times when used with [[ancient DNA]].<ref name=":0">{{Cite journal|last=Bouwman|first=Abigail|last2=Rühli|first2=Frank|date=2016-09-01|title=Archaeogenetics in evolutionary medicine|url=https://link.springer.com/article/10.1007/s00109-016-1438-8|journal=Journal of Molecular Medicine|language=en|volume=94|issue=9|pages=971–977|doi=10.1007/s00109-016-1438-8|issn=0946-2716}}</ref> 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]] <ref name=":3" />, which allows simultaneous amplification and sequencing of all DNA segments in a sample, even when it is highly fragmented and of low concentration<ref name=":0" />. 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. <ref name=":0" /> One method of [[massive parallel sequencing]], developed by Margulies et al., employs bead-based emulsion [[Polymerase chain reaction|PCR]] and [[pyrosequencing]] <ref>{{Cite journal|last=Margulies|first=Marcel|last2=Egholm|first2=Michael|last3=Altman|first3=William E.|last4=Attiya|first4=Said|last5=Bader|first5=Joel S.|last6=Bemben|first6=Lisa A.|last7=Berka|first7=Jan|last8=Braverman|first8=Michael S.|last9=Chen|first9=Yi-Ju|date=2005-09-15|title=Genome sequencing in microfabricated high-density picolitre reactors|url=https://www.ncbi.nlm.nih.gov/pubmed/16056220|journal=Nature|volume=437|issue=7057|pages=376–380|doi=10.1038/nature03959|issn=1476-4687|pmc=PMC1464427|pmid=16056220}}</ref>, 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 <ref name=":1">{{Cite journal|last=Green|first=Richard E.|last2=Krause|first2=Johannes|last3=Ptak|first3=Susan E.|last4=Briggs|first4=Adrian W.|last5=Ronan|first5=Michael T.|last6=Simons|first6=Jan F.|last7=Du|first7=Lei|last8=Egholm|first8=Michael|last9=Rothberg|first9=Jonathan M.|date=2006-11-16|title=Analysis of one million base pairs of Neanderthal DNA|url=https://www.nature.com/nature/journal/v444/n7117/abs/nature05336.html|journal=Nature|language=en|volume=444|issue=7117|pages=330–336|doi=10.1038/nature05336|issn=0028-0836}}</ref>.

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 <ref name=":1" />. This archaeogenetic approach is especially helpful when the [[Morphology (biology)|morphology]] of the fossil is ambiguous <ref name=":2">{{Cite journal|last=Palmer|first=Sarah A.|last2=Smith|first2=Oliver|last3=Allaby|first3=Robin G.|date=2012-01-20|title=The blossoming of plant archaeogenetics|url=http://www.sciencedirect.com/science/article/pii/S094096021100077X|journal=Annals of Anatomy - Anatomischer Anzeiger|series=Special Issue: Ancient DNA|volume=194|issue=1|pages=146–156|doi=10.1016/j.aanat.2011.03.012}}</ref>. Apart from that, species identification can also be done by finding specific [[genetic marker|genetic markers]] in an aDNA sequence. For example, the [[American indigenous people|American indigenous population]] is characterized by specific mitochondrial [[Restriction fragment length polymorphism|RFLPs]] and [[Deletion (genetics)|deletions]] defined by Wallace et al. <ref>{{Cite journal|last=Kolman|first=Connie J.|last2=Tuross|first2=Noreen|date=2000-01-01|title=Ancient DNA analysis of human populations|url=http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1096-8644(200001)111:13.0.CO;2-3/abstract|journal=American Journal of Physical Anthropology|language=en|volume=111|issue=1|pages=5–23|doi=10.1002/(SICI)1096-8644(200001)111:13.0.CO;2-3|issn=1096-8644}}</ref>.

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<ref name=":3" />. The [[Phylogenetic tree|phylogeny]] of some extinct species, such as Australian marsupial wolves and American ground sloths, has been constructed by this method <ref name=":3">{{Cite journal|last=Pääbo|first=Svante|last2=Poinar|first2=Hendrik|last3=Serre|first3=David|last4=Jaenicke-Despres|first4=Viviane|last5=Hebler|first5=Juliane|last6=Rohland|first6=Nadin|last7=Kuch|first7=Melanie|last8=Krause|first8=Johannes|last9=Vigilant|first9=Linda|date=2004|title=Genetic analyses from ancient DNA|url=https://www.ncbi.nlm.nih.gov/pubmed/15568989|journal=Annual Review of Genetics|volume=38|pages=645–679|doi=10.1146/annurev.genet.37.110801.143214|issn=0066-4197|pmid=15568989}}</ref>. [[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<ref name=":3" />.

Another method to investigate relationship between two species is through [[Nucleic acid hybridization|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 [[Nucleic acid hybridization|hybridization]] signal. Scholz et al. conducted [[Southern blot|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 <ref>{{Cite journal|last=Scholz|first=Michael|last2=Bachmann|first2=Lutz|last3=Nicholson|first3=Graeme J.|last4=Bachmann|first4=Jutta|last5=Giddings|first5=Ian|last6=Rüschoff-Thale|first6=Barbara|last7=Czarnetzki|first7=Alfred|last8=Pusch|first8=Carsten M.|date=2000-06-01|title=Genomic Differentiation of Neanderthals and Anatomically Modern Man Allows a Fossil–DNA-Based Classification of Morphologically Indistinguishable Hominid Bones|url=http://www.sciencedirect.com/science/article/pii/S0002929707635447|journal=The American Journal of Human Genetics|volume=66|issue=6|pages=1927–1932|doi=10.1086/302949}}</ref>.

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<ref name=":1" />. 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 <ref name=":1" />. Other similar studies include finding of a mutation associated with dwarfism in ''Arabidopsis'' in ancient Nubian cotton <ref name=":2" />, and investigation on the bitter taste perception locus in Neanderthals <ref>{{Cite journal|last=Lalueza-Fox|first=Carles|last2=Gigli|first2=Elena|last3=Rasilla|first3=Marco de la|last4=Fortea|first4=Javier|last5=Rosas|first5=Antonio|date=2009-08-12|title=Bitter taste perception in Neanderthals through the analysis of the TAS2R38 gene|url=http://rsbl.royalsocietypublishing.org/content/early/2009/08/07/rsbl.2009.0532|journal=Biology Letters|language=en|pages=rsbl20090532|doi=10.1098/rsbl.2009.0532|issn=1744-9561|pmid=19675003}}</ref>.


== References ==
⚫
[[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[11]. This process is repeated several times, and is usually repeated a higher number of times when used with ancient DNA[10]. 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.
{{reflist}}


{{dashboard.wikiedu.org sandbox}}
[[DNA sequencing/Early DNA sequencing methods/High-throughput sequencing (HTP) methods|High-Throughput Sequencing (HTS)]] is an alternative to PCR with a few changes that make it more attractive for use with ancient DNA. HTS involves attaching a generic sequence to every single strand that generic primers can bond to. This makes it so that all of the DNA present is amplified. The DNA is also sequenced as it 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. This allows for the sequencing of highly damaged DNA even when it is present in low concentrations[10].{{dashboard.wikiedu.org sandbox}}

Latest revision as of 05:54, 9 August 2017

Fossil DNA Preservation

[edit]

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[1] and Dense Stereo Reconstruction[2]. Tools used include knives, brushes, and pointed trowels which assist in the removal of fossils from the earth[3].

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[3][4]. Bones are milled to a powder and treated with a solution before the polymerase chain reaction (PCR) process[4]. Samples for DNA amplification may not necessarily be fossil bones. Preserved skin, salt- preserved or air-dried, can also be used in certain situations[5].

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[6].

Methods of DNA Extraction

[edit]

Once a specimen is collected from an archaeological site, DNA can be extracted through a series of processes.[7]  One of the more common methods utilizes silica and takes advantage of polymerase chain reactions in order to collect ancient DNA from bone samples.[8]

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.[9] The best samples to use are fresh fossils as uncareful washing can lead to mold growth.[7] DNA coming from fossils also occasionally contains a compound that inhibits DNA replication.[10] 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.[9]

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.[10] 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.[11]  The general process for extracting DNA using the silica-based method is outlined by the following:[8]

  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.[8]

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.[7] This process is repeated many times, and is usually repeated a higher number of times when used with ancient DNA.[12] 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

[edit]

DNA extracted from fossil remains is primarily sequenced using Massive parallel sequencing [13], which allows simultaneous amplification and sequencing of all DNA segments in a sample, even when it is highly fragmented and of low concentration[12]. 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. [12] One method of massive parallel sequencing, developed by Margulies et al., employs bead-based emulsion PCR and pyrosequencing [14], 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 [15].

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 [15]. This archaeogenetic approach is especially helpful when the morphology of the fossil is ambiguous [16]. 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. [17].

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[13]. The phylogeny of some extinct species, such as Australian marsupial wolves and American ground sloths, has been constructed by this method [13]. 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[13].

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 [18].

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[15]. 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 [15]. Other similar studies include finding of a mutation associated with dwarfism in Arabidopsis in ancient Nubian cotton [16], and investigation on the bitter taste perception locus in Neanderthals [19].

References

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  1. ^ Cohen, David R.; Cohen, Emma J.; Graham, Ian T.; Soares, Georgia G.; Hand, Suzanne J.; Archer, Michael (October 2017). "Geochemical exploration for vertebrate fossils using field portable XRF". Journal of Geochemical Exploration. 181: 1–9. doi:10.1016/j.gexplo.2017.06.012.
  2. ^ Callieri, Marco; Dell'Unto, Nicolo; Dellepiane, Matteo; Scopigno, Roberto; Söderberg, Bengt; Larsson, Lars (2011). "Documentation and Interpretation of an Archeological Excavation: an experience with Dense Stereo Reconstruction tools". [Host publication title missing]. Eurographics Association: 33–40. ISBN 9783905674347.
  3. ^ a b Brothwell, Don R. (1981). Digging Up Bones: The Excavation, Treatment, and Study of Human Skeletal Remains. Cornell University Press. pp. 2–3. ISBN 9780801498756.
  4. ^ a b Scholz, Michael; Bachmann, Lutz; Nicholson, Graeme J.; Bachmann, Jutta; Giddings, Ian; Rüschoff-Thale, Barbara; Czarnetzki, Alfred; Pusch, Carsten M. (2000-06-01). "Genomic Differentiation of Neanderthals and Anatomically Modern Man Allows a Fossil–DNA-Based Classification of Morphologically Indistinguishable Hominid Bones". The American Journal of Human Genetics. 66 (6): 1927–1932. doi:10.1086/302949.
  5. ^ Yang, H.; Golenberg, E. M.; Shoshani, J. (June 1997). "Proboscidean DNA from museum and fossil specimens: an assessment of ancient DNA extraction and amplification techniques". Biochemical Genetics. 35 (5–6): 165–179. ISSN 0006-2928. PMID 9332711.
  6. ^ Scholz, Michael; Bachmann, Lutz; Nicholson, Graeme J.; Bachmann, Jutta; Giddings, Ian; Rüschoff-Thale, Barbara; Czarnetzki, Alfred; Pusch, Carsten M. (2000-06-01). "Genomic Differentiation of Neanderthals and Anatomically Modern Man Allows a Fossil–DNA-Based Classification of Morphologically Indistinguishable Hominid Bones". The American Journal of Human Genetics. 66 (6): 1927–1932. doi:10.1086/302949.
  7. ^ a b c Hagelberg, Erika; Clegg, J. B. (1991-04-22). "Isolation and Characterization of DNA from Archaeological Bone". Proceedings of the Royal Society of London B: Biological Sciences. 244 (1309): 45–50. doi:10.1098/rspb.1991.0049. ISSN 0962-8452. PMID 1677195.
  8. ^ a b c Rohland, Nadin; Hofreiter, Michael (July 2007). "Ancient DNA extraction from bones and teeth". Nature Protocols. 2 (7): 1756–1762. doi:10.1038/nprot.2007.247. ISSN 1754-2189.
  9. ^ a b Handt, O.; Höss, M.; Krings, M.; Pääbo, S. (1994-06-01). "Ancient DNA: Methodological challenges". Experientia. 50 (6): 524–529. doi:10.1007/BF01921720. ISSN 0014-4754.
  10. ^ a b Höss, M; Pääbo, S (1993-08-11). "DNA extraction from Pleistocene bones by a silica-based purification method". Nucleic Acids Research. 21 (16): 3913–3914. ISSN 0305-1048. PMID 8396242.
  11. ^ Yang, Dongya Y.; Eng, Barry; Waye, John S.; Dudar, J. Christopher; Saunders, Shelley R. (1998-04-01). "Improved DNA extraction from ancient bones using silica-based spin columns". American Journal of Physical Anthropology. 105 (4): 539–543. doi:10.1002/(SICI)1096-8644(199804)105:43.0.CO;2-1. ISSN 1096-8644.
  12. ^ a b c Bouwman, Abigail; Rühli, Frank (2016-09-01). "Archaeogenetics in evolutionary medicine". Journal of Molecular Medicine. 94 (9): 971–977. doi:10.1007/s00109-016-1438-8. ISSN 0946-2716.
  13. ^ a b c d Pääbo, Svante; Poinar, Hendrik; Serre, David; Jaenicke-Despres, Viviane; Hebler, Juliane; Rohland, Nadin; Kuch, Melanie; Krause, Johannes; Vigilant, Linda (2004). "Genetic analyses from ancient DNA". Annual Review of Genetics. 38: 645–679. doi:10.1146/annurev.genet.37.110801.143214. ISSN 0066-4197. PMID 15568989.
  14. ^ Margulies, Marcel; Egholm, Michael; Altman, William E.; Attiya, Said; Bader, Joel S.; Bemben, Lisa A.; Berka, Jan; Braverman, Michael S.; Chen, Yi-Ju (2005-09-15). "Genome sequencing in microfabricated high-density picolitre reactors". Nature. 437 (7057): 376–380. doi:10.1038/nature03959. ISSN 1476-4687. PMC 1464427. PMID 16056220.{{cite journal}}: CS1 maint: PMC format (link)
  15. ^ a b c d Green, Richard E.; Krause, Johannes; Ptak, Susan E.; Briggs, Adrian W.; Ronan, Michael T.; Simons, Jan F.; Du, Lei; Egholm, Michael; Rothberg, Jonathan M. (2006-11-16). "Analysis of one million base pairs of Neanderthal DNA". Nature. 444 (7117): 330–336. doi:10.1038/nature05336. ISSN 0028-0836.
  16. ^ a b Palmer, Sarah A.; Smith, Oliver; Allaby, Robin G. (2012-01-20). "The blossoming of plant archaeogenetics". Annals of Anatomy - Anatomischer Anzeiger. Special Issue: Ancient DNA. 194 (1): 146–156. doi:10.1016/j.aanat.2011.03.012.
  17. ^ Kolman, Connie J.; Tuross, Noreen (2000-01-01). "Ancient DNA analysis of human populations". American Journal of Physical Anthropology. 111 (1): 5–23. doi:10.1002/(SICI)1096-8644(200001)111:13.0.CO;2-3. ISSN 1096-8644.
  18. ^ Scholz, Michael; Bachmann, Lutz; Nicholson, Graeme J.; Bachmann, Jutta; Giddings, Ian; Rüschoff-Thale, Barbara; Czarnetzki, Alfred; Pusch, Carsten M. (2000-06-01). "Genomic Differentiation of Neanderthals and Anatomically Modern Man Allows a Fossil–DNA-Based Classification of Morphologically Indistinguishable Hominid Bones". The American Journal of Human Genetics. 66 (6): 1927–1932. doi:10.1086/302949.
  19. ^ Lalueza-Fox, Carles; Gigli, Elena; Rasilla, Marco de la; Fortea, Javier; Rosas, Antonio (2009-08-12). "Bitter taste perception in Neanderthals through the analysis of the TAS2R38 gene". Biology Letters: rsbl20090532. doi:10.1098/rsbl.2009.0532. ISSN 1744-9561. PMID 19675003.
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