Ancient Family Reunion: PCR Reunites Humans to Ancestors

America’s inhabitation by humans is a discussion among archaeologists with new, compelling and contradictory data discovered each year. New methods of research and sources of information sometimes present more questions than answers, but a study has risen to clear some uncertainties – that of Shuká Káa. At first an inconclusive test subject, advanced procedures to amplify Shuká Káa’s ancient deoxyribose nucleic acid (aDNA) illuminated part of our centuries-obscured genomic history.

Genomic research potential expanded following the development of polymerase chain reaction (PCR) analysis for DNA. PCR maximizes whatever tiny source of genetic material is found, perhaps a single hair follicle. The method provides crucial data to bioarchaeology, the study of archaeological human remains. PCR’s laboratory function extends to ancient DNA studies on genome to pathology, population to individual such as Shuká Káa, an ancient hunter gatherer who proved to be an ancient genomic ancestor of Canada’s Pacific Northwest.

DNA as a Genomic Identifier

Discovered twenty years ago in a southeastern Alaskan cave, Shuká Káa was believed to be over 10 thousand years old. He had been exposed to the elements for long enough that archaeologists believed his mitochondrial DNA (mtDNA) would be a more reliable source of genomic data. Shuká Káa’s mtDNA was extracted and sequenced to determine his lineage among other ancient individuals (Kennewick Man and Anzick-1) as well as contemporary North Americans.

mtDNA is a separate sequence with hundreds of copies per cell. It consists of around 17,000 pairs to the 30 billion in nDNA. Mitochondria are inherited along the matrilineal line, so mtDNA carried by an individual imitates their biological mother’s. mtDNA has lower ID resolution, but it’s useful in degraded samples due to its longevity in destructive conditions.

Contained in mtDNA are SNPs along with areas known as hypervariable regions (HV1 and HV2) which have been observed as highly mutable. Single nucleotide polymorphism (SNPs) manifest variable DNA nucleotides at one loci. These portions of DNA don’t code for phenotypes, yet genetic studies treat them as alleles from which identity can be assessed.

Unfortunately, in the case of Shuká Káa, no direct haplogroup matches were found, and he was reburied in 2008 in the ceremonial tradition of indigenous groups. The mystery of his relation to contemporary tribes remained, but scientists had not exhausted their research on his identity.

DNA has a projected stability of up to one million years, but no samples are discovered under the conditions necessary for ideal preservation. DNA’s code is obscured by centuries of environmental degradation, so more work is necessary to extract it. In cases like Shuká Káa’s when the sequence is damaged, fractured or otherwise too miniscule to guarantee testing accuracy, portions of DNA are amplified by PCR.

PCR Methods with aDNA

Amplifying DNA is as essential in aDNA investigation as it is in biochemistry. “Ancient DNA” is a classification of nDNA and mtDNA without defined boundaries of age: all historical, archaeological or otherwise ancient specimens are relegated to this category.

Like the DNA used in forensic or biochemical tests, aDNA must be identified and isolated from cellular samples. This is more challenging in ancient remains like Shuká Káa’s due to varying levels of degradation. Archaeological human sources of DNA include bones, dentition and hair follicles. Such tissues are ideal for PCR because they take a longer period than soft tissue does to decay, so even heavily decomposed or mummified samples are viable for replication purposes.

Old samples are endangered by their extended exposure to unmediated environments. Basic decomposition, acidity, temperature fluctuations, moisture, bacteria, and even the contents of air will contribute to DNA degradation. If a sample isn’t adequately prepared, the integrity of sequencing tests are risked. Conclusive data is therefore achieved through DNA amplification by PCR. If enough aDNA can be salvaged, amplification begins until the number of replicates is conducive to sequencing.

There is a specific approach of PCR more precise and better suited for aDNA. Taq polymerase is the enzyme commonly employed for PCR replication, responsible for the attaching nucleotides to a template of DNA. Multiple displacement amplification (MDA) replaces Taq polymerase with ɸ29 DNA polymerase derived from the bacteriophage ɸ29. It amplifies DNA in a mechanism similar to the rolling circle method. The results have a lower rate of error and come in fractions averaging 10kb, larger than those produced in Taq-mediated PCR. Allelic dropout and preferential amplification have still been observed, so the method is not infallible, but it is an advantage for bioarchaeologists.

Shuká Káa’s aDNA was sequenced again in 2017 with PCR amplification and admixture analysis. This time, with methodology 10 years further matured, nDNA was sampled from the tissue of his molars. Only 6% of his genome was recovered due to advanced DNA damage. Even with a fraction of his genome, this new data reinitiated the search for Shuká Káa’s descendants.

Applying PCR to aDNA Studies

aDNA studies have a place in our contemporary culture. The early years of PCR were witness to the exhumation of a skeleton reported to be Auschwitz’s infamous “Angel of Death,” Dr. Josef Mengele. Highly degraded samples were taken from the femur, and microsatellite alleles were amplified. Comparison to Mengele’s son confirmed paternity and gave strong evidence to the skeleton’s identity. A 2014 PCR on mtDNA identified Aaron Kominski as iconic killer Jack the Ripper over a century after his infamous crimes occurred. Skepticism persists, because the research was conducted proceeding a nonfiction book entailing the results.

Bioarchaeological aDNA goes further than a couple of centuries. The field surveys archaeological remains to understand individual, population and environmental experiences through human history. Two focal aspects in the expertise include genomic and pathological studies of ancient people. Ancient human DNA research is lucrative to both trajectories; environmental interactions are revealed with the incorporation of aDNA from other organisms. PCR is an indispensable tool for bioarchaeologists to unlock the ancient world, and the key to such knowledge is in individuals like Shuká Káa.

HUMAN GENOME

Instead of having genomes from only contemporary individuals, ancient people have been rediscovered with PCR. Often, only fractions of a genome can be extracted. These can be amplified to give insights into an ancient person’s genetic attributes by examining specific loci for individualizing characteristics.

Whole genomic amplification (WGA) is another route which studies an individual’s comprehensive genome. This method produces a robust sample of genomic aDNA. PCR-based WGA methods use Taq polymerase to gather a range of the represented genome in 3kb fragments. The alternative isothermal method, MDA, binds random hexameters to denatured DNA strands using ɸ29 polymerase, producing up to 99% genome coverage.

Biological relatedness or biodistance, the focus of Shuká Káa’s case study, is analyzed via PCR-visualized genomic variation. Biodistance is defined in bioarchaeology as a measure of relatedness among groups divided by temporal and geographical factors. Genetic traits reflecting these differences are unveiled by aDNA surveys. Researchers infer from biodistance what kinship, social striations or genetic disorders differentiated the population. PCR employed for this purpose therefore assists in reviewing relatedness, intra-/inter-population relationships, population history and more.

Genomic population studies range from archaic hominins to post-colonization in the Americas. Here are just a few notable examples:

• The Denisovan group of Siberia, human ancestors who contributed 4-6% of their genome to contemporary Melanesians, received 1.9 fold coverage from a single phalange and tooth. From these miniscule mtDNA samples, a model of population history and lineage identified Denisovan divergence from Neanderthals.
• mtDNA from 24 Neolithic agriculturalists 7500 years old brought context to European ancestry. In analysis, researchers determined Neolithic farmers had a low genetic influence on modern female lineages of mtDNA in comparison to Paleolithic hunter-gatherers.
• 50 individuals of a pre-Colombian Amerindian group on Norris Farms were analyzed with PCR methodology. Based on mtDNA, four lineages were determined, and the results indicated these were the major groups present during colonization.

Genomic projections can be individual but have been conducted on scales for entire species. The Human Genome Project benefited from PCR and electrophoresis testing. Another genome sequence was conducted for Neanderthals: three individuals provided 4 billion nucleotides, giving context to their gene flow and influence on modern human populations in Afro-Eurasia.

Research on Shuká Káa’s genome compared the new nDNA sequence to dental samples of indigenous groups and British Columbian individuals from 6075-1750 years ago. The results were surprising: his genome was closely associated to the British Columbian skeletons, and these were related to a number of indigenous Pacific Northwest groups in haplogroup D4h3a and A2. His mtDNA and nDNA consequently suggested kinship to the Tsimshian, Tlingit, Nisga’a, and Haida tribes. It was inferred Shuká Káa is a common ancestor and the region has genetic continuity for at least ten millennia.

Broader implications also surfaced. Shuká Káa was proven genetically dissimilar from the nearly 13,000 year old Montanan Anzick Child and Kennewick Man. This data implied that, at this period in the Americas, at least two genetically distinct lineages existed. Experts thus concluded that the initial inhabitation of the Americas is more complex than previously hypothesized.

HUMAN PATHOLOGY

Biological relatedness can also be interpreted from pathology, and PCR helps bioarchaeologists evaluate ancient disease identifiable in human remains. It recognizes known genetic characteristics for hereditary disorders and detects the relics of infectious disease. Disease - inherited or contracted - is visualized to understand frequency and impact. Those which still afflict contemporary populations like tuberculosis and syphilis have extra significance, because understanding their origins and past influence can aid in current experimentation.

Genetic disorders are easily located at their known loci. Infection is not so easily identified in archaeological circumstances. Certain diseases infest a population without leaving physiological impressions. Of these pathogens, though, some leave genetic indicators in those they’ve infected.

One aDNA experiment searched for the mycobacterium tuberculosis complex (MTBC) in 133 geographically and temporally spaced skeletal samples. Four quantitative PCR assays were conducted to locate ancient MTBC indicators at conserved regions of the complex. They targeted the rpoB gene and insertion elements IS6110 and IS1081 believed to be specific to MTBC complex indication. Seven samples were positively identified with probe technology.

Leprosy (Mycobacterium leprae) is a disease historically coexisting with tuberculosis. More common as an epidemic in ancient times, it’s characterized by long-term incubation and neurological complications. Leprosy was one of 92 pathogens featured in a study that parallel-screened archival samples of bone, dental pulp and mummified ancient tissue. Pathogenic strains were isolated in their genomic conservation sequences, and ancient M. leprae DNA was located in the predetermined sample. The efficiency of this screening technique was an added achievement to the amplification technique.

Some vectored plagues leave no skeletal signature due to their rapid killing power, but they can be detected in aDNA. Malaria epidemics (Plasmodium falciparum) and bubonic plagues (Yersinia pestis) have changed human immunology and impacted the genome of affected societies. Parasitic malaria DNA was found in ancient Roman samples using hybridization capture mtDNA of modern strains. Similar PCR results were produced with the Yersinia pestis specific plasminogen activator gene: “plague pit” remains in the Netherlands, England and France have revealed aDNA of the bubonic pathogen.

ENVIRONMENT

Other experiments focus on ancient species and environments which influenced our evolution. While not autonomous from human bodies, paleomicrobiology – studying the microbiome of ancient humans – is genetically explored through the analysis of dental calculus. One approach to the oral microbiome is amplicon sequencing the variable regions V1-V9 in the highly conserved bacterial 16S rRNA gene. This method, of course, requires amplification which can be achieved with the primer-based tactics of PCR. Scientists thus know more about the diet, behavior and microbes of ancient life.

Investigating domesticated species of ancient human agriculture is integral to chronicling the human condition. Archaegenomic examination uses DNA techniques previously developed for human study on botanical samples. The crop aDNA is sequenced, and short-term genome evolution is inferred to be caused by selective manipulated by humans in adaptation to drought. Such rapid change was documented in cotton from Africa, Brazil and Peru as well as barley in Qasr Ibrim, Egypt. aDNA therefore reveals historical behavior of humans interacting with domesticated species.

A final class of research involves what is known as “environmental” DNA (eDNA). Environmental samples are collected from water, ice and sediments. They contextualize biodiversity in a region for thousands of years; DNA from flora and fauna are also analyzed for transitions and extinctions. Some organismal DNA can be located in the sediment itself, as aDNA of extinct woolly mammoths and moa birds were found in Siberia and New Zealand. aDNA from organisms persists if the environmental matrix contains large organic molecules, particles or minerals to protect it from nuclease activity. Other aDNA is conserved through natural transformation when other cells integrate extracellular DNA into their genomes.

Future Research with PCR

The discoveries of aDNA, mtDNA, and PCR have coalesced to give us valuable insight into the past of our species, and it can be inferred that the benefits of this scientific industry will proliferate in the future. Sequencing the genome of Shuká Káa is only one example of our ever-advancing scientific endeavors. The subject of America’s first inhabitants has not yet been concluded, but we are nearing the answers which have evaded previous research.

Laboratories worldwide have become involved in the inspection of DNA far older than the science itself. Kits are dispensed to researchers interested in accessing the genome of damaged samples; the supplies for analysis include ladders for genotyping, PCR mix, primer mix, and positive control DNA. Using the same electrophoresis PCR as is conducted on known samples, aDNA researchers bring life back to material long dead and almost lost in the degrading circumstances of time.

New technology is being developed to make PCR more efficient: rapid ID, bead hybridization binding to SNPs, SNP scanning chip hybridization and SNP-STR parallel sequencing are just a few new resources rising in the field. Next Generation Sequencing (NGS) also offers promising results beyond the current projection of PCR. With the addition of newly developed technology, it’s inevitable that even more discoveries will be made from retrospectively studying our ancestors globally.

[Agarose LE, DNA 1kb ladder, DNA 100bp ladder]

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Megan Hardie GoldBio Staff Writer

Megan Hardie is an undergraduate student at The Ohio State University’s Honors Arts and Sciences program. Her eclectic interests have led to a rather unwieldly degree title: BS in Anthropological Sciences and BA English Creative Writing, Forensics Minor. She aspires to a PhD in Forensic Anthropology and MA in English. In her career, she endeavors to apply the qualities of literature to the scientific mode and vice versa, integrating analysis with artistic expression.

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