Report on the DNA analysis from skeletal remains from two skulls
conduced by Trace Genetics

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 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

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

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

additional PCR inhibitors co-extracted with the DNA, the DNA solution was purified using the

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:

1. The sample was run in parallel with a reagent blank (negative control) but was not

processed with any other samples.

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

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

4. The sample was left in EDTA with agitation for 22 days prior to digestion with

proteinase-K.

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

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:

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

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

3. To ensure adequate demineralization of the sample, the sample was left immersed in

EDTA with agitation for 30 days.

4. The final elution from the silica spin columns (Promega®) was performed twice, each

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.

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:

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

2. The sample was immersed in EDTA for 26 days prior to digestion with proteinase-K.

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].

The sample taken from the Starchild Skull (SCS-1) has mtDNA consistent with Native

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.

AMPLIFICATION METHODS

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

Taq DNA polymerase (Gibco) 2 to 6 μl of DNA template and sterile ddH20 sufficient to bring

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

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

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

and clothing whenever possible; 3. Use of separate pre- and post-PCR facilities; 4. Periodic UV

irradiation and bleaching of all materials used to help eliminate any surface contamination; 5.

Running negative controls at all stages of the extraction and amplification process to identify the

presence of contaminants; 6. 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.

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