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Scientific Testing - Ancient DNA Test

The Starchild’s mitochondrial DNA was relatively easy to recover and showed it had a human mother (expected if it is an alien hybrid), but its nuclear DNA, the part that would reveal its father’s genetic heritage, couldn’t be recovered with current primers. We were advised to wait for primers to become more efficient. We were also advised to investigate its bone chemistry because in conducting the DNA tests, some intriguing discoveries were made.

1. The bone was significantly harder to cut that it should have been.

2. There was a stronger-than-usual smell of "burning bone" when cutting it.

3. When put into EDTA, the normal solvent for human bone, the Starchild should have dissolved in a week or less since it is less than half as thick as normal human bone. 10 weeks later the Starchild bone had not dissolved a bit.

4. When a strong detergent known as ’tween 20’ was added to the mix, the Starchild bone dissolved completely, overnight, down to a thin layer of residue. Unexpected.

Thus, its chemistry seemed to be unusual enough to warrant a full-scale investigation. For most of 2004 we did precisely that, and now we have some astonishing results that have scientific merit and investigative significance of the highest magnitude.

 


 


 

 

Analysis

 

 

The main analysis so far is contained in 3 reports:

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PRELIMINARY ANALYSIS OF A HIGHLY UNUSUAL HUMAN-LIKE SKULL
Dr. Ted J. Robinson

M.D., L.M.C.C., F.R.C.S (c)


The skull in question has a provenance that is not verified at present. That situation may change in time, but for now all that can be said with certainty is that the skull is real, it is comprised of calcium hydroxyapatite (the essence of all mammalian bone), its parts are configured "naturally" (not cobbled together or in any other way hoaxed), and it presents numerous physical anomalies that do not conform to standard skull norms.

The skull remained in my possession in Vancouver, B.C., for the better part of one year. I was given complete discretion to study it in any way I saw fit. My analysis derives from extensive examination of the skull itself, combined with analysis of X-rays and CAT scans. I have shared these data with colleagues who have given opinions that will be mentioned in this document as their input becomes relevant.

In general, the skull has the basic components of a human skull: i.e., a frontal bone, two sphenoids, two temporals, two parietals, and an occipital. However, these bones have been markedly reconfigured from the "normal" shapes and positions such bones usually have. In addition, the bone itself has been reconstituted to an equally marked degree, being somewhat less than half as thick as normal human bone, with a corresponding weight of roughly half normal. The reconfigurations and the reconstitution are uniform throughout all axes and in all planes of the skull. There is no asymmetrical warping or irregular thinning that is the hallmark of typical human deformity.

The morphology of this skull is so highly unusual as to be unique in my forty years of experience as a medical doctor specializing in plastic and reconstructive surgery of the cranium. Because of its uniqueness, I undertook an extensive review of current literature on craniofacial abnormalities, which failed to uncover a single similar example. In short, it seems to be not only unique in my personal experience, but also unique throughout the past history of worldwide study of craniofacial abnormalities. This is significant.

Specialists who examined the skull and associated X-rays and CAT scans were:

  • Dr. Fred Smith, Head of Pediatrics, Children’s Hospital, New Orleans, La.

  • Dr. David Hodges, Radiologist, Royal Columbian Hospital, New Westminster, B.C.

  • Dr. John Bachynsky, Radiologist, New Westminster, B.C.

  • Dr. Ken Poskitt, Pediatric Neuroradiologist, Vancouver Children’s Hospital

  • Dr. Ian Jackson, (formerly of Mayo Clinic), Craniofacial Plastic Surgeon, Michigan

  • Dr. John McNicoll, Craniofacial Plastic Surgeon, Seattle

  • Dr. Mike Kaburda, Oral Surgeon, New Westminster, B.C.

  • Dr. Tony Townsend, Ophthalmologist, Vancouver

  • Dr. Hugh Parsons, Ophthalmologist, Vancouver

  • Dr David Sweet, Forensic Odontologist, Vancouver

Dr David Hodges, a radiologist, stated that the suture lines were open and growing at the time of death. Dr.David Sweet, an internationally renowned forensic pathologist at the University of British Columbia, was of the opinion that the skull was that of a 5-6 year old, based upon the dentition in the right maxillary fragment[1].

Though some specialists who looked at the skull disagreed, I have always supported Dr Sweet in his belief that this was the skull of a 5-6 year old child.

Dr. Bachynsky noted that there is no evidence of erosion of the inner table of the skull. Such erosion would be consistent with a diagnosis of hydrocephaly, so this condition can safely be ruled out as a cause of the abnormalities expressed. Hydrocephaly also causes a widening of the sutures, again not expressed here. There was consensus agreement to both of these observations by other experts conversant with these features.

Dr. Kaburda carried out special three-dimensional X-rays which measure certain fixed points in any skull, allowing for comparison of any particular skull to the established norm. These accumulated results were compared to a statistical analysis of 100 human skulls. This skull was found to be more than ten (10) standard deviations outside the norm, i.e. the statistical center of a Bell curve. This is another strong indication that the skull in question is unlike anything previously seen or investigated.

Doctors Townsend and Parsons examined the orbital cavities and concluded that the being may well have been sighted, but if so, its visual structures deviated strongly from the norm. The cavities, while astonishingly symmetrical, were less than 50% normal depth. The optic foramen, which carries the optic nerve from the brain through the orbital bone to the eye, is nearly an inch lower than it would be in a normal human skull. However, attachment points for the muscles that control an eyeball’s movements were still to be felt on the inner surface of the orbit, indicating that a ball rather than some other mechanism was its most likely expression.

If indeed these sockets held eyeballs, those of normal size would have greatly protruded from the face, creating a serious liability of damage during routine activity. Because the eyeballs occupy a position lower in the face than is normal, and they rest in a socket markedly reduced in rectilinear shape and depth, they would have been significantly reduced in size. In either case, however, large eyeballs or small, they would require upper lids three or four times more extensive than normal upper lids to be lubricated in the manner necessary for human eyeballs to function properly.

Doctors Hodges and Poskitt found the brain inside the skull was abnormally large. This was determined by lining the intracranial cavity with a plastic bag that was then filled with Niger birdseed. This gave a size of 1600 cubic centimetres, which is 200 c.c. larger than the typical adult size of 1400 c.c. This is even more unusual because the size of the skull compares most favourably with a small adult or a child of about 12 years old. This extra brain capacity is apparently due to the deep shallowing of the eye sockets, a total lack of frontal sinuses (not even vestigial bumps are discernable), and significant bossing (expansion) of the upper rear of both parietals.

In any case, they observed, the extreme slant of the rear parietals and the occipital bone challenges whether this skull could have contained typical brain matter, and casts further doubt that its cerebellum was typical. In a normal skull, the cerebellum rests at the base of the cerebrum, supported by the internal occipital protuberance and the twin flares of the sagittal sulcus and the transverse sulcus. With this support mechanism, over the course of a lifetime the cerebrum’s weight does not press down onto the cerebellum and distend it such that it will cease to function properly. In this unique skull, however, the entire weight of the brain slants directly down on the area that should hold its cerebellum. Instead of the rounded area typically present for support, there is a wedge-shaped area of perhaps one-quarter of normal. Furthermore, the internal protuberance and sulcus ridges are significantly reduced. What effect would the weight of a notably amplified brain have on an unsupported cerebellum carried into adulthood? It presents a genuine conundrum.

Personally, I was most concerned with determining how the rear of the skull could have become so flattened, from the atypical fossa (depression) in the sagittal suture between the parietals, down to the foramen magnum opening. This could not have been caused by any kind of flattening or binding device because the surface of the occipital reveals the subtle convolutions inevitably present in unaltered skulls. Skulls that undergo any kind of shaping technique will always reveal such technique with a distortion of the bone surface. Lacking even a hint of evidence of shaping, and of any unnatural or premature fusing of any sutures, it is entirely safe to say that the extreme flattening of the skull was caused by its natural growth pattern and is not artificial. This too is significant.

Another of my concerns is that the external occipital protuberance (inion) is absent from its notable position in the center of the occipital bone, and indeed is represented by an actual slight fossa (depression) in the surface. (As mentioned earlier, the same is true for its internal counterpart, which has been greatly reduced.) It seems clear that the neck of this being attached to its skull much lower than in a normal skull, centered under the balance point for both lateral and medial flexion. Even more unusual, the neck itself seems to have a circumference somewhere in the range of 50% of usual neck volume, which presents yet another example of the thorough uniqueness of this specimen.

In addition to lacking frontal sinuses, there is no sign of the brow ridges evident in normal skulls. Its upper orbits are thin edged rather than rounded. Its zygomatic arches are greatly reduced and significantly lowered from their usual positions. Its mastoid processes are less than normal, as are all connective points for the lower face (which would attach to the coronoid process and condylar process of the missing mandible). Based on these observations, its lower face may have been as much as 50% reduced from normal. On the other hand, its inner ears are noticeably larger than normal, again pushing into the range of 50% larger. This is also true for the condyles abutting the spinal atlas.

A detached upper right maxilla contains two molars [recent note: one has been lost to testing]. Tooth wear on the molars indicates maturity was reached, yet another set of teeth are present in the maxilla and appear ready to take the place of those mature teeth when and if they are lost or are no longer useful. The question of age at death remains open.

Carbon 14 Dating has shown the Human Skull to be 900 years old ± 40 years[2].

These and other mysteries about this skull await further analysis by other experts wishing to help determine its origin and history.

 

 

References:

[1] Dr Matthew Brown, a Dentist in London, made close-up x-rays images of the maxilla in September 2004. He states that the roots of unerupted teeth are consistent with those of a child who was about 4˝ yrs old.

[2] Carbon 14 dating was also carried out on Starchild Skull Bone in July/August 2004 which produced the same result - 900 years old ± 40 years

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Report on Maxilla and Teeth X-Rays
Dr Matthew Brown, DDS.

In September 2004, some New X-rays of the Starchild Teeth were completed. The report and results are shown below.
 

27 October 2004

Examination of infantile right maxilla, anterior fragment, with regard to dentition.

Visual:

One tooth present (upper right first deciduous molar (54*)). Alveoli (sockets) of upper right first deciduous incisor, second deciduous incisor, deciduous canine and deciduous second molar present (51, 52, 53, 55). Anterior/mesial wall of upper right permanent first molar (16) crypt visible. The crown of 54 shows occlusal wear facets and enamel lamellae.

Radiographic.

 

Crowns of upper right permanent

. central incisor (11)
. lateral incisor (12)
. canine (13)
. first premolar (14)
. second premolar (15)

visible, with initial root development on the central incisor; crowns of the remaining teeth at an incomplete stage of development.

Interpretation:

The absent teeth 51, 52, 53, 55, and 16 were lost at post- or peri-mortem. The development of the unerupted permanent teeth suggests an age of 4.5 - 5 years.

*F.D.I. notation
 


X-Rays are Shown in "Positive" and "Negative" Views

 

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Report from Trace Genetics

 

 

 

 

Report on the DNA analysis from skeletal remains from two skulls August 12, 2003


Two sets of remains were received by Trace Genetics and were processed for genetic analyses. The remains consisted of two skulls presented by Mr. Lloyd Pye for DNA analysis.


SAMPLING
Prior to attempts to extract DNA from the remains, the remains were inventoried and taped using a video camera. Video records of the sampling procedure and the initial extraction on all samples were taken and archived by Trace Genetics.


Samples were cut from the left parietal of an abnormally shaped skull, identified as the Starchild skull on February 10, 2003. Equipment used to sample was sterilized using a bleach solution prior to use. Sampling was performed in a room not used for any genetic analyses. Fragments weighing a total of 0.8g were cut from the parietal using a rotary cutter with a previously unused blade. The fragments were placed in a sterile conical tube labeled SCS-1 and stored for analysis. A second 0.7g fragment adjacent to the sample retained by Trace Genetics was placed in a sterile conical tube labeled SCS-2 and returned to Mr. Pye.


Two teeth were removed from maxilla of a skull presented in association with the Starchild skull on February 10, 2003. The right first molar tooth and root weighing 1.7g was removed and labeled “SA-1.” The tooth and root were placed in a sterile conical tube and retained for genetic analysis by Trace Genetics. A portion of the root was fractured in the process and remained in the maxilla. The right premolar and root (sample labeled “SA-2”; total weight 1.0g) were also removed from the maxilla, placed in a sterile conical tube. The SA-2 sample was returned to Mr. Pye.

EXTRACTION AND ANALYSIS OF DNA SCS-1:

Extraction 1:
A first extraction was performed on a 0.24g fragment of the parietal bone from sample SCS-1. The extraction was performed in a dedicated ancient DNA laboratory beginning on 7 March 2003 and was performed in parallel to an extraction of SA-1 and a reagent blank (negative control). Both surfaces of bone were sanded with a rotary sander to remove any surface contaminants and lacquer preserves present on the outer surfaces of bone. Subsequent to sanding, the bone was exposed to ultraviolet (UV) irradiation (254nm) for 300 seconds per side. The bone surface was then cleaned with bleach (2% sodium hypochlorite), rinsed with sterile EDTA and placed in a fresh 15ml conical tube and immersed in approximately 2ml of 0.5M EDTA. The tube was sealed with parafilm and placed on a rocker.


After 10 days, the tube was opened and 150µl of 0.1M PTB [1] and 20µl of 100mg/ml proteinase-K was added to the sample and EDTA. The sample was incubated with agitation overnight at 64oC. DNA was extracted from the digested sample using a 3-step phenol/chloroform extraction method. Two extractions with phenol:chloroform:isoamyl


(25:24:1) of equal volume to the digested product were followed by an extraction with an equal volume of chloroform:isoamyl (24:1). The extracted DNA solution was concentrated by ammonium-acetate precipitation using two volumes of cold 100% filtered ethanol and 1/2 volume of 5M ammonium acetate. This solution was then stored at -20�C for approximately 4 hours to facilitate precipitation, then centrifuged at high speeds (10,000-12,000rpm) for 15 minutes to pellet the precipitated DNA. The supernatant was discarded and the remaining DNA pellet dried and resuspended in ~300µl sterile ddH2O. To further purify the DNA and remove additional PCR inhibitors co-extracted with the DNA, the DNA solution was purified using the Promega� Wizard PCR Preps DNA Purification Kit as directed by the manufacturer. DNA was eluted from Promega� columns with 100µl sterile ddH2O, the elutant labeled SCSex1 and stored at –20�C.


Attempts to amplify segments of mtDNA from extract SCSex1 were performed as described below in METHODS. Single amplifications for fragments containing the diagnostic mutations for Native American haplogroups A, B, C and D[2] did not reveal a known Native American haplogroup, however, the extraction did not amplify consistently. A single amplification of a fragment of the mtDNA first hypervariable segment (HVSI) between np 16210 and np 16328 was sequenced using a cycle sequencing procedure with ABI Big-Dye 3.1 chemistry and analyzed on an ABI automated genetic analyzer. The sequence obtained revealed a transition relative to the Cambridge reference sequence at np16273. This sequence did not match either any personnel with access to the ancient DNA facilities or a sequence obtained from Mr. Pye. Subsequent amplifications of this fragment were not successful and the sequence could not be confirmed. Attempts to amplify fragments of the amelogenin gene located on the X and Y chromosome[3] were uniformly not successful.

Extraction 2:
A second extraction was performed beginning April 21, 2003 on 0.21g of the parietal sample from SCS-1. The extraction was performed as above with the following modifications:

  • The sample was run in parallel with a reagent blank (negative control) but was not processed with any other samples.
     

  • The bone was exposed to 900 seconds of UV irradiation per side.
     

  • The bone was completely immersed in 2% sodium hypochlorite for 5 minutes.
     

  • The sample was left in EDTA with agitation for 22 days prior to digestion with proteinase-K.
     

  • At digestion, ~50µl of Tween-20 was added with 100µl of PTB.
     

  • The silica extraction columns (Promega®) were eluted with 80µl of ddH2O and sample labeled “SCSe2.”

Attempts to amplify mtDNA for fragments containing the diagnostic mutations for Native American haplogroups A, B, C and D were performed on extract SCSe2. Multiple amplifications indicated that the sample possessed an AluI restriction site at np 13262 indicative of Native American haplogroup C [2]. Sequence obtained for a fragment of the first hypervariable segment of the mtDNA control region from np16210 to np 16367 revealed transitions at np16223, np 16298, np 16325 and np 16327. These mutations are characteristic of haplogroup C in the Americas [4].

Multiple attempts to amplify a segment of the amelogenin gene were unsuccessful using various amounts of SCSe2 extract as template. 30µl of the original extract was concentrated to a final volume of ~10µl using a microcon YM-30 concentrator. Attempts to amplify this concentrated template were not successful.


Extraction 3:
A third extraction was performed beginning on June 4, 2003 as described above for extraction 2 with the following modifications:

  • The extraction was performed on the entire remaining 0.40g of bone.
     

  • The sample was immersed in ~3.5ml of EDTA.
     

  • To ensure adequate demineralization of the sample, the sample was left immersed in EDTA with agitation for 30 days.
     

  • The final elution from the silica spin columns (Promega®) was performed twice, each time with 35µl of ddH2O preheated to 65oC.

Attempts to amplify fragments of mtDNA were performed to test for the presence of diagnostic mutations fo r Native American haplogroups A and C. The sample did not appear to possess the diagnostic HaeIII mutation and np663 indicative of haplogroup A. Multiple amplifications did reveal the presence of the AluI site gain at np13262 indicative of haplogroup C.


A single amplification of a fragment of the amelogenin gene located on the X and Y chromosomes [3] produced a single amplification product 106bp in length. Multiple subsequent amplifications did not reproduce this event, as all subsequent attempts did not produce a PCR product.

SA-1:

Extraction 1:
A first extraction was performed on 0.53g fragment of the molar tooth from sample SA-1 beginning on 7 March 2003 and was performed in parallel to an extraction of SCS-1 (above) and a reagent blank (negative control). The extraction was performed in the manner describe above for extraction 1 of SCS-1 save that the outer surface of the tooth, which had previous to sampling been firmly rooted in the maxilla, was not sanded and the final elution of the silica spin column (Promega®) was eluted to 100µl and labeled SAex1.


Multiple attempts to amplify segments of mtDNA containing amplifications for fragments of mtDNA containing the diagnostic mutations for Native American haplogroup A revealed a HaeIII restriction site at np663 consistent with known Native American haplogroup A [2]. Amplifications for fragments containing the diagnostic sites for haplogroups B, C and D did not show presence of mutations indicative of these haplogroups. A single amplification of a fragment of the mtDNA first hypervariable segment (HVSI) between np 16210 and np 16327 revealed transitions relative to the Cambridge reference sequence at np16223, np16290 and np16319. These mutations are consistent with Native American haplogroup A.


Multiple amplifications of a fragment of the amelogenin gene on the X and Y chromosomes consistently produced a single band 106bp in length when visualized on an electrophoretic gel consistent with DNA from a female [3].

Extraction 2:
A second extraction was performed beginning Ap ril 21, 2003 on 0.42g of the tooth sample from SA-1. The extraction was performed similar to extraction 1 on SA-1 (above) with the following modifications:

  • The sample was not run in parallel to any samples from SCS-1.
     

  • The sample was immersed in EDTA for 26 days prior to digestion with proteinase-K. The final elution was labeled SAe2 and stored at –20oC.

Multiple amplifications of a mtDNA fragment indicated the presence of a HaeIII restriction site at np663 indicative of Native American haplogroup A. Amplifications of the extraction did not possess the AluI site gain at np16262. Multiple amplifications of a fragment of the amelogenin gene produced a single band when visualized on an electrophoretic gel consistent with DNA from a female.

DISCUSSION:
MtDNA from virtually all modern, full-blooded Native Americans belongs to one of five mitochondrial lineages or matrilines (designated haplogroups A, B, C, D, and X) marked by the presence or absence of characteristic restriction sites or by the presence of a nine base pair (9-bp) deletion [2, 5]. Analyses of ancient DNA from Native Americans likewise indicates that these haplogroups constitute virtually all prehistoric Native American individuals as well [see: 6].

American haplogroup C, as revealed through two independent extractions performed on fragments of parietal bone. While a single first extraction did not appear to type similarly, this inconsistent result is likely a product of a low level of contamination. This single extraction neither amplified consistently nor was the single sequence of HVSI reproducible. Contamination could have occurred either prior to sampling, introduced in the extraction process, or during PCR amplifications. It is unlikely that contamination could account for the haplogroup C mtDNA as this type is not possessed by any researcher with access to the ancient DNA facilities and the reagent blanks did not indicate systematic contamination in the extractions.


The sample taken from the associated skull (SA-1) has mtDNA consistent with Native American haplogroup A as determined through both extractions. The sample also appeared be from a female individual as evidenced by repeated amelogenin typing. It is unlikely that contamination could account for the haplogroup A mtDNA as this type is not possessed by any researcher with access to the ancient DNA facilities and the reagent blanks did not indicate systematic contamination in the extractions.


As mtDNA exists in high copy number (upwards of three orders of magnitude relative to any single copy nuclear DNA locus), it can be recovered from prehistoric biological material in sufficient quantities for amplification and analysis using the polymerase chain reaction (PCR) [see: 7, 8]. MtDNA is present in haploid condition with inheritance being passed down exclusively through maternal lines [9]. Thus, that the samples analyzed from SCS-1 and SA-1 possessed markedly different mtDNA types excludes a mother-offspring relationship between the two individuals. As it was possible to type and confirm both to known pre-Columbian mtDNA types found in the Americas, both individuals most appear to have possessed Native American mothers.

While it is possible to obtain nuclear DNA as well from ancient samples, the reduced copy-number at any particular nuclear locus relative to mtDNA makes it less likely that a particular extract will contain sufficient DNA for the analysis of a nuclear genetic locus using presently available PCR methods. The ability to amplify nuclear DNA from the SA-1 extractions but not from the SCS-1 extractions could be a product of any of a number of factors. In ancient DNA analysis, success rates from teeth are generally higher than from bone [10, 11]. Further, there is some indication that X-Ray exposure damages and degrades DNA, which may have decreased the quantity and quality of DNA available in the bone prior to extraction.

 

The lone amplification using the amelogenin primers on extract SCSe3 could not be confirmed through additional amplifications and likely indicates a sporadic contamination of a single PCR reaction caused either by a female individual in the laboratory or could have been introduced to laboratory disposables (e.g. pipette tips, PCR reaction tubes). Such contamination has been noted elsewhere [12] and consequently, any conclusions drawn from the single un-reproduced PCR reaction should not be taken as any reliable indication as to the DNA present in the sample.


The presence of reliably typed mtDNA from SCS samples does indicate that mtDNA is present in the bone. The inability to analyze nuclear DNA indicates that such DNA is either not present or present in sufficiently low copy number to prevent PCR analysis using methods available at the present time.

All reagents used to extract and amplify were first tested to detect any DNA contamination and ancient DNA facilities were cleaned using bleach to remove possible sources of contamination. Further additional contamination controls and precautions are described below.


PCR amplifications of mtDNA were conducted in 25 µl volumes using 4µl dNTPs (10mM), 2.5µl 10X PCR buffer (Gibco), 1.3µl BSA (20mg/ml), 0.75µl MgCl2, 0.2µl Platinum Taq DNA polymerase (Gibco) 2 to 6 µl of DNA template and sterile ddH20 sufficient to bring reaction volume to 25µl. After an initial 4-minute denaturation step at 94°C to activate the hot start Taq, 40 PCR cycles were performed consisting of a 94oC denaturing step, a 50-55°C annealing step (temperature depending on primers utilized), and a 72°C extension step of 30 seconds each. A final 3-minute extension at 72°C was added after the last cycle. A portion of the amplification product (~5µl) was run on a 6% polyacrylamide gel together with a size standard ladder, stained with ethidium bromide and photographed under UV light using a digital imager (ISO 2000 imaging system, Alpha Innotech, San Leandro, CA).

 

To assess the presence or absence of diagnostic restriction sites, the remaining 20µl were incubated with 10 units of the appropriate restriction enzyme overnight at 37o C, and then subjected to electrophoresis in the manner previously described. Primers used for amplification of these segments and restriction enzymes used are shown in table 1.


Amelogenin amplifications [3] were attempted using 1, 3, and 8 µl of DNA template in 25 µl reaction volumes, adjusting ddH2O amounts to maintain concentrations of other reagents.
 

Site First Primer Coordinate Second Primer Coordinate Polymorphism Reference:
Haplogroup A 591-611 765-743 HaeIII np663 “+” = A [13]
Haplogroup B 8195-8215 8316-8297 9 base pair deletion Deletion = B [14]
Haplogroup C 13236-13257 13310-13290 AluI np13262 “+” = C [15]
Haplogroup D 5099-5120 5211-5190 AluI np 5176 “-“ = D [15, 16]
HVSI 16192-16209 16385-16368 Sequence [15, 17]
HVSI 16192-16209 16348-16328 Sequence [15, 17]


CONTAMINATION CONTROLS:
Ancient DNA is typically highly degraded and survives in much lower copy numbers than modern DNA. Consequently, ancient DNA is highly vulnerable to contamination from modern sources and specific precautions against contamination, as summarized by Kelman and Kelman [18], were utilized in this study to both minimize contamination and, importantly, to identify contamination when present so that it does not lead to false inferences.

 

These measures include:

1. Use of dedicated laboratory space, supplies, reagents and equipment for preparation of ancient DNA samples inside UV irradiated glove boxes;

2. Use of sterile, disposable labware irradiation and bleaching of all materials used to help eliminate any surface contamination;

3. Running negative controls at all stages of the extraction and amplification process to identify the presence of contaminants;

4. Confirmation of results by multiple amplifications of multiple extractions. All positive results were confirmed though multiple amplifications of each extraction and multiple extractions performed at different times.



References Cited

  • Poinar, H.N., et al., Molecular coproscopy: Dung and diet of the extinct ground sloth Nothrotheriops shastensis. Science (Washington D C), 1998. 281(5375): p. 402-406.
     

  • Schurr, T.G., et al., Amerindian mitochondrial DNAs have rare Asian mutations at high frequencies, suggesting they derived from four primary maternal lineages. American Journal of Human Genetics, 1990. 46(3): p. 613-23.
     

  • Mannucci, A., et al., Forensic Application of a Rapid and Quantitative DNA Sex Test by Amplification of the X-Y Homologous Gene Amelogenin. International Journal of Legal Medicine, 1994. 106(4): p. 190-193.
     

  • Torroni, A., et al., Asian affinities and continental radiation of the four founding Native American mtDNAs. American Journal of Human Genetics, 1993. 53(3): p. 563-590.
     

  • Forster, P., et al., Origin and evolution of native American mDNA variation: A reappraisal. American Journal of Human Genetics, 1996. 59(4): p. 935-945.
     

  • Eshleman, J.A., R.S. Malhi, and D.G. Smith, Mitochondrial DNA Studies of Native Americans: Conceptions and Misconceptions of the Population Prehistory of the Americas. Evolutionary Anthropology, 2003. 12(1): p. 7-18.
     

  • Kaestle, F.A. and D.G. Smith, Ancient mitochondrial DNA evidence for prehistoric population movement: The Numic expansion. American Journal of Physical Anthropology, 2001. 115(1): p. 1-12.
     

  • Stone, A.C. and M. Stoneking, mtDNA analysis of a prehistoric Oneota population: Implications for the peopling of the New World. American Journal of Human Genetics, 1998. 62: p. 1153-1170.
     

  • Giles, R.E., et al., Maternal Inheritance of human mitochondrial DNA. Proceedings of the National Academy of Sciences of the United States of America, 1980. 77(11): p. 6715-6719.
     

  • Malhi, R.S., Investigating prehistoric population movements in North America with ancient and modern mtDNA, in Anthropology. 2001, University of California: Davis.
     

  • Hummel, S., Ancient DNA Typing: methods, strategies and applications. 2003, Berlin: Springer-Verlag.
     

  • Schmidt, T., S. Hummel, and B. Herrmann, Evidence of contamination in PCR laboratory disposables. Naturwissenschaften, 1995. 82(9): p. 423-431.
     

  • Stone, A.C. and M. Stoneking, Ancient DNA from a pre-Columbian Amerindian population. American Journal of Physical Anthropology, 1993. 92(4): p. 463-471.
     

  • Wrischnik, L.A., et al., Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA. Nucleic Acids Research, 1987. 15: p. 529542.
     

  • Handt, O., et al., The retrieval of ancient human DNA sequences. American Journal of Human Genetics, 1996. 59(2): p. 368-376.
     

  • Parr, R.L., S.W. Carlyle, and D.H. O'Rourke, Ancient DNA analysis of Fremont Amerindians of the Great Salt Lake Wetlands. American Journal of Physical Anthropology, 1996. 99(4): p. 507-518.
     

  • Eshleman, J.A., Mitochondrial DNA and prehistoric population movements in Western North America, in Anthropology. 2002, University of California, Davis: Davis, CA.
     

  • Kelman, L.M. and Z. Kelman, The use of ancient DNA in paleontological studies. Journal of Vertebrate Paleontology, 1999. 19(1): p. 8-20.

 

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