Research:

Ancient DNA from the beginning

       Ancient DNA (aDNA) refers to DNA recovered from clinical, museum, archeological, and paleontological specimens. aDNA ranges in age from less than 100 yrs to tens of millions of yrs. The study of aDNA is a young field, which has been revolutionized by the application of PCR technology, and interest is growing rapidly. Disciplines as diverse as evolution, anthropology, medicine, agriculture, and forensic science have all been influenced by the analysis of aDNA.

     That aDNA could be extracted and characterized was first demonstrated in non-human material in 1984 by Higuchi and colleagues, who identified nucleic acids from a museum specimen of the extinct quagga and showed its phylogenetic affinity to modern zebra. A year later Paabo 1985 obtained DNA sequence data from a 2400 yrs old Egyptian mummy. This result was surprising not only for its demonstration of the remarkable antiquity for which molecular genetic analysis was apparently possible, but also for the large DNA fragments sequenced (>3kb).

      Following the early enthusiasm for aDNA research came fundamental observations on the nature of aDNA preservation, high failure rate of amplification of many samples, and concerns regarding the authenticity of aDNA samples. Nucleic acids of any antiquity are degraded and modified in various ways. Because of the degraded nature of aDNA extracts, mitochondrial DNA (mtDNA) has proven to be the molecule of preference for genetically characterizing prehistoric samples. This is because mtDNA is present in several hundreds of copies per cell, in contrast to the single-copy nuclear genome. Thus, target sequences of mtDNA are more likely to be present in any single extract, and accessible for amplification, than are nuclear sequences. However, in some well-preserved samples, nuclear markers have been screened, and several methods for molecular determination of sex have been developed. Additionally, genomic DNA has been used to confirm the presence of diseases organisms in prehistoric samples.

Post-mortem DNA decay:

         When an organism dies, its DNA normally becomes degraded by endogenous nucleases. Under fortunate circumstances, such as rapid desiccation, low temperatures or high salt concentrations, nucleases can them selves become destroyed or inactivated before all nucleic acids are reduced to mononucleotides. If this is the case, slower but still relentless processes start affecting the DNA. For example, oxidation, as well as the direct and indirect effects of background radiation, will modify the nitrous bases and the sugar-phosphate backbone of the DNA. Furthermore, deamination, depurination and other hydrolytic processes will lead to destabilization and breaks in DNA molecules. All these processes create problems for the retrieval of ancient DNA sequences. For example, a high proportion of cytosine and thymine residues in extracts of ancient tissues are oxidatively modified to HYDANTOINS (Oxidation products of the Pyrimidine bases (Cytosine and Thymine)), which block DNA polymerases and thus the PCR1. Furthermore, deamination products of cytosine, for example, are common in ancient DNA and cause incorrect bases to be inserted during the PCR2.

        After a long enough time, the cumulative effects of damage to the DNA will become so extensive that no useful molecules remain. Assuming physiological salt concentrations, neutral pH and a temperature of 15 °C, it would take about 100,000 years for hydrolytic damage to destroy all DNA that could reasonably be retrieved. Some environmental conditions, such as lower temperatures will extend this time limit, whereas other conditions will reduce it. However, to the best of our understanding, to consider amplification of DNA molecules older than one million years of age are overly optimistic.

Biochemistry of aDNA:

      There are two principal obstacles to the recovery of aDNA. Molecular degradation limits the amount of amplifiable DNA available and organic PCR inhibitors often coextract with the DNA. Nucleic acids gradually degrade over time through processes such as hydrolysis and oxidation. Hydrolysis is the breakdown of the N-glycosyl bond between the sugar and the base in the presence of water.

     Guanine and adenine are 20_fold more susceptible to removal (depurination) than cytosine or thymine (depyrimidination ), although the rate is temperature and PH dependent. Conversely, hydrolytic deamination of the bases affects pyrimidines (30,000 years half-life in vivo ) at a rate 40-fold higher than the purines . Oxidation is the process by which water-derived hydroxyl or superoxide radicals modify bases or distort the helix. Because mitochondria are the center of O2 metabolism, oxidation primarily affects the mitochondrial rather than nuclear genome. Hydantions (oxidized pyrimidines) suspected to do the most damage to DNA. Their presence negatively correlated with success in extraction and amplification of aDNA, most likely due to the fact that they block extension during PCR. These degradation processes occur continuously in vivo (100_500 times cell^-1 day^-1), but in the nucleus are under stringent control by DNA repair mechanisms. Postmortem, these and other degradative events continue to accumulate. Such alterations to aDNA molecules have been detected by high _pressure liquid chromatography and electron microscopy. For these reason, recovery and amplification of aDNA, when possible, is usually limited to fragments <300_500 bp in length, and only for samples in the range of tens of thousand, or fewer, years old. A 20C decrease in temperature reduces base degradation 10-to 25-fold. It is not surprising, then, that Paabo and colleagues observe an inverse correlation (nonsignificant) between long-term environmental temperature and DNA extraction and amplification success. This inverse correlation is also observed in our laboratory when comparing extraction and amplification success rates of prehistoric populations under study from the North American Arctic, the Great Basin, and the US southwest.

Tuross (1994) reports an inverse correlation between sample age and total DNA yield. However, because this was assessed electrophoretically by comparative ethidium bromide staining, it may also reflect coextracting bacterial and /or fungal DNA. Tuross further suggests that at least for bone samples, the breakdown of DNA primarily occurs immediately postmortem, most likely because of the stabilization of DNA binding hydroxyapatite, which slows the hydrolytic depurination rate twofold. Environmental factors such as temperature (4-37 C), humidity (20%-98%), PH (3.0-10.0), exposure to seawater, and burial in garden soil or sand does not significantly affect DNA yields from forensic dental samples.

Because aDNA recovery is destructive process, defining suitable candidates for aDNA extraction and amplification attempts is of considerable import. Nucleic acid degradation can be monitored several ways. Amino acid racemization, the transformation of L-into D-enantiomers (tow optical isomers of amino acids), like depurination of DNA, is also affected by temperature and the presence of water. The rate of aspartic acid (ASP) racemization is approximately equal to that of DNA depurination and, therefore, is a good predictor of DNA preservation. Poinar et al (1996) report they could retrieve and amplify DNA from samples in which the Asp L-form by >= 9%. Another method, gas chromatography /mass spectrometry; can be used to measure the relative amounts of modified DNA bases.

The majority of extracted PCR inhibitors are tannins, humic acids, and fulvic acids, all common soil –derived degradation products. Because they are highly phenolic, they generally should be removed by phenol–chloform extraction. Another class of inhibitors are maillard products, by –products of sugar reduction, which cross –link macromolecules, including nucleic acids. Humic acids, fulvic acids, and maillard products often result in brown coloring of DNA extracts. These compounds often fluoresce blue in agarose gel under ultraviolet (UV) light.

Controlling for contamination:

        Due to the sensitivity of the PCR and the degraded nature of DNA in ancient samples, the contamination of samples and laboratory preparations by exogenous DNA is a constant concern. Such contamination can derive from a variety of sources, including the DNA of other workers who have handled the samples before they reach the laboratory, such as archaeologists, museum staff, and medical workers. Additionally, some relatively standard procedures for dealing with skeletal remains can serve to either worsen contamination problems, or degrade the endogenous DNA. For example, washing samples in water can facilitate the infiltration of contaminating DNA deep into the bone matrix, rending more difficult the decontamination process. Further, x-raying bones can increase the fragmentation of the endogenous DNA. Several decontamination procedures (see blow) are employed in an attempt to remove contaminating surface DNA from samples before beginning the extraction protocol.

     Exogenous DNA can also be introduced into samples from a variety of other sources. A substantial source of contaminating DNA can be the modern DNA extracted in laboratories for other purposes, as well as the DNA that has been PCR amplified for analysis. Consequently, the laboratories in which aDNA analysis is performed must be physically separated from other laboratories conducting molecular analysis, and must be dedicated solely to the extraction and analysis of DNA from ancient samples. Additionally, workers cannot move from laboratories in which modern and post-PCR work is conducted directly into the aDNA laboratories because of the high probability of transporting modern or amplified DNA is a particularly high risk associated with moving from other laboratories, it remains a risk at all times. Therefore, the use of protective clothing is necessary. A combination of laboratory coats, coveralls with hoods, hairnets, shoe covers, gloves, and facemasks proves effective. Plasticware and reagents used in the process of DNA extraction. The most effective strategy to minimize the changes of contamination via this route is to purchase both reagents and disposable plasticware that are guaranteed to be DNA-free by the manufacture. Additionally, reagents should be aliquoted into small volumes that will be used quickly to avoid the introduction of DNA to stock solutions. Finally, laboratory surfaces need to be maintained to prevent the accumulation of DNA. Regularly wiping surfaces with bleach, and subjecting them to periods of UV- irradiation, can achieve this.

      Even when all the precautions described are followed, contamination is an inevitable reality of working with aDNA. Recognizing that contamination will occur necessitates the ability to identify it when it does. Negative controls are run in parallel with samples throughout the extraction procedure, in which empty tubes are treated in exactly the same manner as the tubes containing samples. If the products of these negative controls yield amplifiable DNA, it is apparent that the extraction has been contaminated.  A negative control of the PCR reaction is also run, to assist in determining at which point in the procedure the contamination is at the level of analysis. DNA sequences obtained from ancient samples should be phylogenetically sensible.

Authenticity of Ancient DNA:

Authenticity of aDNA is of paramount concern, and efforts to assure that research results reflect endogenous target sequences rather than modern contaminations have received considerable attention (Handt et al 1994, Richard et al 1995). Handt (1994) recommend six criteria for evaluating authenticity of aDNA result:

1- Pre- and post-PCR activities should be spatially separated in the lab, or performed in different laboratories.

2- Strict laboratory protocols should be adopted to prevent and monitor the introduction of modern DNA.

3- Controls should be used routinely to monitor contamination.

4- Replicate samples should be used to confirm initial results.

5- Observed aDNA sequence data should make phylogenetic sense, and,

6- An inverse relationship between fragment size and PCR efficiency should be observed.

PCR inhibition:

Co-extracted PCR inhibitors can be a substantial problem in working with aDNA. Employing the silica GuSCN protocol in preference to the phenol-chloroform protocol can eliminate some inhibitory problems, but inhibitors are frequently found to be in extracts despite using this protocol. Additional strategies include diluting the DNA extract in hope that the inhibitory elements will be sufficiently diluted for successful amplification, before the target DNA is diluted to such a degree that it is no longer amplifiable. Further, bovine serum albumin (BSA) can be added to the PCR reaction, which can serve to bind to inhibitors, thereby removing them from solution and allowing the reaction to proceed. Other strategies include further digesting the samples with proteinase K or a collagenase, or adding NaOH. 

aDNA and sex determination:

The most common method of genetic sexing takes advantage of differences in the Amelogenin gene, present on both the Y and X chromosome, but with slightly varying sequences. The favored protocol involves amplifying a short segment of the Amelogenin gene that contains a 6-base-pair (bp) deletion in the copy on the X chromosome, when compared with the Y chromosome. Thus the DNA fragment amplified from an X chromosome is only 106 bp long, while that from a Y chromosome is 112 bp. Amplifications from a male individual will therefore contain DNA fragments of two sizes, while those from a female individual will contain DNA fragments of only one size (sex determination markers).

Paleoparasitology:

Paleoparasitology may be developed as a new tool to parasite evolution studies. DNA sequences dated thousand years ago, recovered from archaeological material, means the possibility to study para-site-host relationship coevolution through time. Together with tracing parasite-host dispersion throughout the populations, Paleoparasitology points to the interesting field of evolution at the molecular level. Also this may help in elucidating the possible role of parasites in shaping both the genetic and natural history of human populations.  

aDNA and the infectious disease:

aDNA techniques have also been applied to questions of patterns of prehistoric disease. Various infectious diseases like TB and HIV can leave similar skeletal pathologies on human remains, and indeed infectious diseases can manifest in patterns indistinguishable from each other.

Ancient DNA in Sudan and the great expectations

      The use of ancient DNA (aDNA) in the reconstruction of population origins and evolution is becoming increasingly common. The resultant increase in number of samples and polymorphic sites assayed and the number of studies published may give the impression that all technological hurdles associated with aDNA technology have been overcome. However, analysis of aDNA is still plagued by two issues that emerged at the advent of aDNA technology, namely the inability to amplify a significant number of samples and the contamination of samples with modern DNA.

      Sudan is the largest country in Africa as well as one of the most diverse, with 90 discrete ethnical groups represented, and over 130 languages spoken. However, despite long-term investigations into the history and prehistory of this country and its people, the origins of this diversity remain to a large extent poorly understood. Evidence for the history of the Sudan is extremely limited before the 1800s with the major exception of the Nile Valley. We know quite a lot about the Kerma, Napata, Meroe and Nile Nubian civilizations although we do not understand Meroitic and are not sure of its relationship to other present-day language groups of the Sudan or Africa. Reliable scholars have rejected the possibility of Meroitic being related to Afro-Asiatic languages (the nearest geographically is Beja). Recent attempts to review the situation incline towards it being part of Nilo-Saharan, but this is still unproven.

Ancient DNA is young field in Sudan started since 2002 in Institute of Endemic Diseases with collaboration of Sudan National Museum, and this project is going to cover different historic periods in Sudan including Neolithic, Meroitic, post-meroitic, Christian and Islamic era.

objectives:

1. To identify and characterize the skeletal remains found in burial sites in Sudan by anthropometrics measures and ancient DNA analysis, and to design the phylogenetic tree of extant Sudanese populations.

2. Also this project will detect the presence of Mycobacterium tuberculosis, Leishmania and Malaria parasites from ancient human remains. Initially ancient DNA will be extracted from bone samples. According to the PCR results that will be obtained, the hereditary and genetic information of the organism will be explored and characterized using a molecular based approach. 

Report on the Nuri mummy

Length: Unmeasured

Sex: Male

Age: Unknown
According to our interest we received some specimens from ribs & skin of Nuri Mummy (March2002) from Sudan National Museum to extract Ancient DNA (aDNA) and to determine the origin of this mummy.
Two methods were used for aDNA extraction, phenol/chloroform &silica methods. The extraction result showed excellent DNA quantity (40.2ng/ul), and a dark brown solution was also recovered which may be from the high concentration of Melanin (the person was obviously quite dark skinned) or some chemicals used during mummification if there was any. The quality and quantity of DNA might be an indication of a relatively recent age of the body (No > than 600 years), although exception might take place here depending on the condition of preservation.
Polymerase chain reaction (PCR) was used to amplify aDNA with YAP primers a Y chromosome marker which have recently been recognized as useful as a  population specific markers. The mummified body YAP profile is positive indicating that he is most likely to affiliate to ethnic groups west of the Nile in accordance with preliminary dada from African populations. Also it is an indication of a 5% chance only for him to be of the Nilotic group who are usually YAP negative.

We should also note that the success of the PCR reaction itself might indicate absence of some inhibitory chemicals.

Few insect pupae were recovered from the body and were sent to Dr. ElTigani Alam from the Natural history Museum for identification. Preliminary results indicate that they are flesh flies a known source of myiasis and infestation of decomposing  bodies. It indicate that the body might have been exposed for some time  following death, as part of a pre-Islamic funeral rite.

Conclusion:
According to the above results, Nuri Mummy is male& with an excellent quantity of aDNA, from a pre-Islamic period. Unfortunately neither age determination nor Carbon dating were carried out.
The brown material resulting from the extraction should be analyzed to give information about composition of this chemicals.
This finding is very good chance to start aDNA studies in Sudan to know about human populations.

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Y chromosome analysis of Nuri Mummy.  Lane 1 negative control,   lane2 &3 YAP+, lane 4 100bp DNA marker. The products were run on 2% Agarose gel electrophoresis

Hisham Y. Hassan                                            Dr. Muntaser E. Ibrahim

Date: 8/2/2003