Date Published: October , 2009
Publisher: A.I. Gordeyev
Author(s): A.P. Grigorenko, S.A. Borinskaya, N.K. Yankovsky, E.I. Rogaev.
Studies of ancient DNA specimens started 25 years ago. At that time short mitochondrial DNA (mtDNA) fragments were the main targets in ancient DNA studies. The last three years were especially productive in the development of new methods of DNA purification and analysis. Complete mtDNA molecules and relatively large fragments of nuclear DNA are the targets of ancient DNA studies today. Ancient DNA studies allowed us to study organisms that went extinct more than ten thousand years ago, to reconstruct their phenotypic traits and evolution. Ancient DNA analyses can help understand the development of ancient human populations and how they migrated. A new evolutionary hypothesis and reconstruction of the biota history have been re-created from recent ancient DNA data. Some peculiarities and problems specific to the study of ancient DNA were revealed, such as very limited amounts of DNA available for study, the short length of the DNA fragments, breaks and chemical modifications in DNA molecules that result in “postmortem” mutations or complete blockage of DNA replication in vitro. The same specific features of DNA analysis were revealed for specimens from complicated forensic cases that result in the lack of experimental data or interpretation problems..
Here, we list the specific features of ancient DNA methodology and describe some achievements in fundamental and applied research of ancient DNA, including our own work in the field.
Ancient DNA studies allow to empirically verify evolutionary hypotheses and contribute to the complex reconstruction of historical changes in biota. The analysis of DNA from human archeological samples reveals information on the genetic traits of ancient inhabitants of various geographical regions.
Paleontological and archeological materials and biological samples that are collected at excavation sites or stored in museums yield very small amounts of DNA that is usually highly fragmented. Moreover, this ancient DNA is modified in various ways that prevents amplification or lead to errors in nucleotide sequence reads. Because of the low efficiency of amplification of authentic DNA extracted from ancient and historic samples, contamination of the sample by even a single modern DNA molecule can produce errors. A number of specific measures must be taken in order to prevent contamination and to detect possible contamination. False positive results, caused by in-lab contamination, are one of the major problems in ancient DNA studies. That is why the key step in molecular-genetic analysis of ancient and historic samples is DNA extraction.
Postmortem DNA alterations, and mutations during in vitro DNA amplification, are among the central methodological problems in ancient DNA and complex forensic DNA analysis. As opposed to metabolically active tissues that have an active DNA reparation system postmortem cells accumulate chemical (hydrolytic or oxidative) DNA modifications and strand damage. Studies show that postmortem DNA damage includes strand breaks, loss of bases and cross-linking between strands that inhibits PCR. Postmortem alterations that modify bases but do not inhibit amplification are especially important, since they can cause the appearance in the amplification products nucleotide of changes that were not present in the authentic sequence (type I substitutions A > G / T > C and type II substitutions C > T / G > A) (Table 2). The manner how the degraded DNA templates are damaged depends on the samples age, their geographic origin, and the taphonomic conditions (preservation conditions) of the environment where the samples were stored. Postmortem alterations can appear in mutational hot-spots, thus simulating evolutionary mechanisms . The manner and dynamics of accumulation of postmortem DNA alterations are under continuous research [38, 39]. DNA damage limits the size of the DNA fragments found in ancient samples to about 100-500 bp. That is why the primers for ancient DNA PCR are usually chosen for no more than 200-300 bp fragments, although fragments of greater length have been obtained in some cases (Fig. 2).
Ancient DNA analysis involves sequencing of a large number of short fragments that have multiple overlapping of the same genomic regions. Low sequencing speed and high cost limit the usage of such research. Novel technologies of massively parallel sequencing of a large amount of DNA samples have appeared in the last 3-4 years, and the cost has dropped by two orders of magnitude. This novel technologies have given researchers sequencing possibilities that were previously available only to large genomic centers. Among the available novel technologies several were used in ancient DNA studies, such as clonal amplification followed by parallel sequencing of dense micropanels of cloned DNA fragments by repeated enzymatic reaction cycles, with automatic registration of the signal from each cycle and every fragment.
The technological approaches applied for ancient DNA study can also be used for forensic genetic analysis in difficult cases where only micro-scopic amounts of material are available or the DNA has been severely damaged. Some of these approaches were used in the genetic expertise of the putative remains of the family of the last Russian Emperor Nicholas II Romanov. In the early 1990s, a first grave with human remains was found near Yekaterinburg. During the investigation, it was suggested that the remains belong to the family of the Russian Emperor Nicholas II Romanov, his wife, the Empress Alexandra Fedorovna, their 3 daughters, the court physician, and three servants. They are all thought to have been murdered in 1918 [42-44].
They are all thought to have been murdered in 1918 [42-44]. However, the remains of two children of the Romanov family were not identified, and their fate remained unknown. Among other hypotheses, there has been a legend that Alexey and Anastasia, the youngest children of the Romanov family, had survived those turbulent times. In July 2007, a second grave was found not far from the first one.
It contained burned bone fragments from two skeletons. Forty-four bone fragments were found in the second grave, all severely damaged by fire and presumably sulfuric acid. Preliminary anthropological analysis of the half-burned bone fragments from the second grave suggested that the bones belonged to a boy 10-14 years of age and a young woman of about 18-23. The least damaged fragments of the femoral bones from both the male and female skeletons were selected for genetic analysis, and they were labeled Samples 146 and 147, respectively. Samples from the first grave were also collected for a more detailed study, and reference samples were taken from living relatives of Nicholas Romanov and Alexandra Fedorovna. Furthermore, swabs of blood stains from a shirt that had belonged to Nicholas II and is stored in the Hermitage museum were also used for analysis. The study included the following steps: preparation of the samples for DNA extraction; DNA extraction; quantification of the extracted total DNA and human-specific DNA; amplification and sequencing of the mitochondrial hyper-variable regions, and later sequencing and reconstruction of the complete mtDNA (cmtDNA) sequence; determination of the STR-profiles of the Y-chromosome; determination of the autosomal STR-profiles; additional sex identification with the use of a special marker designed for degraded DNA analysis [45, 46]; and extraction and analysis of modern DNA from Romanov family relatives and their comparison to historic samples. The steps and methods of DNA identification are described in Table 3 .
Table 3Methodical approaches specific to analyses of degraded DNA from historical specimens Stage of analysisSpecial proceduresReagents and methodsPreparation of historic samplesIndependent analysis in specialized laboratories in IOGene (Moscow) and University of Massachusetts Medical School (Worchester, USA).Physical and chemical cleaning of small bone fragments, crushing or drilling to obtain bone powder.Extraction of DNA from bone remainsAll the experimental procedures were performed in sterile PCR-hoods, in accordance with standards for ancient DNA research, keeping to all the safety precautions so as not to contaminate the samples by modern DNA. DNA was extracted from ~170≤700 mg of cleaned bone material treated by 0.5 M EDTA and proteinase K and was then purified by a QIAquick PCR purification kit (Qiagen) in accordance with the manufacturer’s protocol with slight modifications.Extraction of DNA from archive spots of blood The biological material was obtained from 4 different blood stains. At least 3 swabs were taken from each spot. In order to minimize contamination, DNA was extracted only from the 2nd and 3rd swabs of each spot.DNA was extracted with the QIAamp DNA Mini Kit (Qiagen) in accordance with the manufacturer’s protocol (“DNA Purification from Dried Blood Spots”) with several modifications.Quantative DNA analysisThe total DNA was quantified by the Quant-iT™ PicoGreen® Assay kit (Invitrogen), human specific DNA was quantified by the Plexor® HY assay kit (Promega) and the 7500 Real-Time PCR System (Applied Biosystems).Sequencing HVR1 and HVR2 of mtDNA from historic samples Possible contamination by foreign DNA was monitored by using negative controls (amplification of “empty” extracts and PCR without addition of the template).mtDNA fragments were amplified as short overlapping fragments. The PCR products were then extracted from the agarose gel using a QIAquick Gel Extraction kit or a MinElute Gel Extraction kit. For additional studies, the PCR products of samples from the second burial site were cloned.Sequence analysis of the complete mitochondrial genome, extracted from bone remains.Since the DNA was highly degraded, short overlapping fragments sized 164-383 b.p. were obtained, covering the whole mitochondrial genome. Because the amount of DNA was initially so small, multiplex amplification was performed using 88 pairs of specially developed primers grouped into 3 kits, and then the products of this PCR were amplified with individual primer pairs. The PCR products were then sequenced using three different strategies Analysis of the mtDNA extracted from the blood stains on Nicholas the Second’s shirt.Up to 5 or 7 repeated PCR reactions were conducted for some of the SNPs.Since the quality of preservation in the blood stains was unknown, a kit of primers was developed for the amplification of short (64≤109 b.p.) DNA fragments, which would include very rare SNPs identified in the previous analysis of Skeleton №4 (the putative skeleton of Nicholas the Second).Extraction and analysis of DNA from modern samples.All the procedures involved in analyzing modern DNA were performed in other buildings, which were located some distance away from the ancient DNA laboratories. All the living relatives who took part in the study gave their written consent.DNA obtained from buccal swabs or drops of blood was extracted using standard protocols. PCR was performed using a kit of primers for amplifying longer fragments.Assembly of fragments into a continuous sequence of nucleotides (contigs).The sequences were assembled using Seqman software, DNASTAR, and the revised Cambridge reference sequence (rCRS, accession number AC_000021) as a standard.Sex identification.Sex was identified using the standard method, amplification of a fragment of the amelogenin gene using several commercial kits: AmpF≤STR® MiniFiler™ (Applied Biosystems) and PowerPlex S5 (Promega). Specially developed primers for the amplification of short fragments specific to the X- and Y-chromosomes were also used. Analysis of nuclear STR markers.During the initial study, mtDNA or nuclear DNA extracts that consisted of a mix of individual profiles were discarded from further analysis. Each sample from various extracts was serially amplified. Homozygous loci were considered authentic if multiple independent amplifications confirmed a certain allele for the autosome STR-marker. The following kits were used in order to obtain autosomal STR profiles from bone samples of the first and second burial sites: AmpF≤STR® MiniFiler™ PCR Amplification Kit (Applied Biosystems) and PowerPlex S5 System (Promega), developed especially for analyzing degraded DNA.STR-profiles of the Y chromosome.The AmpF≤STR® Yfiler™ (Applied Biosystems) kit was used, according to the manufacturer’s protocol with slight modifications for work with degraded DNA.Electrophoresis analysis In order to increase the signal intensity and lower “noise” in the STR-profiles, the products of multiplex amplification were sometimes purified according to the method for genotyping low-copy DNA templates using Qiagen MiniElute PCR purification kit. Electrophoretic analysis was performed with a 96-capillary sequencer 3730xl DNA Analyzer (Applied Biosystems). The results were analyzed using GeneMapper ® ID software v3.2 (Applied Biosystems).
Complete nucleotide sequences of the mitochondrial genome have been determined for the putative remains of Nicholas II and Alexandra Fedorovna from the first grave; and the putative remains of Alexey and his sister, from the second grave. The mitotypes of the putative remains of Nicholas II and Alexandra Fedorovna are from the common European mtDNA haplogroups T2 and H1.
To study the paternal lineage DNA profiles of the putative remains of Emperor Nicholas II and Prince Alexey, the STR-haplotypes of the Y-chromosome were determined. Specialized procedures were developed in order to increase the PCR sensitivity, since the amount of the available DNA was limited, and the molecules were highly fragmented (some of the methods are described in Table 3) . The STR-profiles were determined from multiple independent PCR amplifications using no less than three different DNA extracts. Only the alleles that were identified in no less than 2 amplifications were considered authentic. A full Y-STR profile for the bone specimen of Skeleton №4 and for the museum samples of Nicholas II’s blood was obtained using these criteria. Low-copy highly fragmented DNA often loses single STR alleles. Marker DYS385 shows two loci on the Y-chromosome. The high molecular weight allele (DYS385/ 14) was identified only once in the repeated experiments with the DNA extracted from Sample #146, thus this allele for Sample #146 is indicated as not determined (ND). DNA isolated from the archival Nicholas II bloodstain and DNA obtained from Romanov paternal lineage family members were used as reference samples (Fig. 6). Y-chromosome STR-profiles of the studied samples and the reference sequences were completely identical (Fig. 7 and Table 5). This 17-locus Y-STR haplotype is unique. It is not found in large population databases for multi-locus Y-STR (Table 4) and was first encountered in the described study .
There is historical evidence that Prince Alexey suffered from severe bleeding that is characteristic of hemophilia. It is now known that hemophilia is caused by insufficient activity of blood clotting factors. Factor VIII deficit caused by mutations in the F8 gene is the cause of the most common hemophilia A (about one in 5 000 boys is born with this disease), and Factor IX deficit causes hemophilia B (F9 gene), which occurs 5 times less often.
The methods of DNA analysis developed and widely used in recent years allow experimental identification and reconstruction of DNA nucleotide sequences extracted from biological samples preserved for prolonged periods of time in natural conditions or have been subjected to DNA and its con-stituent molecules damage. The possibility of a successful study of such samples is provided by the use of novel sequencing strategies, novel methods for extracting and purifying DNA, specially equipped facilities, variety of control experiments, and additional methods of data analysis that prevent interpretation errors in the data. All this factors have contributed to the extraction of genetic information from organisms that have disappeared tens of thousands of years ago, and to the reconstruction of evolutionary events, which was hitherto unachievable in experimental study. These findings opened new possibilities for precise molecular genetic analysis of severely damaged and decayed DNA, which has already raised the standard of ap-plied procedures in forensic medicine. The results reviewed in this paper could not have been obtained without the development of novel DNA tech-nologies that can now be incorporated into the everyday routines of fundamental and applied research, making them more reliable, fast and informa-tive, as well as lowering costs.
The study was supported by the Russian Federal Agency for Science and Innovation (federal contract 02.512.11.2231) and by the Program “Biodiversity” of Presidium of Russian Academy of Sciences.