The document discusses the author's ancestry tracing results through mitochondrial DNA (mtDNA) testing. Three tests were performed - on Hypervariable Region I, Hypervariable Region II, and a coding region single nucleotide polymorphism backbone test. The results identified the author's mtDNA haplogroup as I, which is found in approximately 2% of Europeans and traces back to a group called the Aurignacians that inhabited Europe approximately 30,000-40,000 years ago. The document provides background information on mtDNA, including its organization, mutation rate, and inheritance solely through the maternal line.

1. Ancestry Tracing With mtDNA
Organizational Diagram of Circular Mitochondrial DNA.
Note: every graphic in this paper is sourced from http://www.genebase.com/index.php
maintained by Genetrack
Lou Sheehan, Biology 215
July 28, 2008
Professor Dee M. Walter

2. ABSTRACT
This paper discusses the testing for the mtDNA haplogroup of one individual. The tests were
of Hypervariable Region I, Hypervariable Region II and of the Coding Region via an SNP
Backbone Test. The paper includes supporting discussions of the identified haplogroup – I –
and the nature of mitochondrial DNA.
INTRODUCTION
DNA provides a record of our biological past. The translation of this – if one will –
history book advances daily. One chapter in this book details our lineage.
Earlier in 2008 the author’s mitochondrial DNA (henceforth as: “mtDNA”) was tested.1
The Tests involved
(i) Hypervariable Regions I,
(ii) Hypervariable Region II, and an
(iii) mtDNA Single Nucleotide Polymorphism Marker (“SNP”) Backbone Test.
All testing was performed by Genetrack.
This paper will generally discuss the above-mentioned three tests. The results showed
the author’s mtDNA haplogroup to be “I”. There are 82
major haplogroups in Europe: H
(47%), J (17%), U (11%), T (9%), K (6%), X (6%), V (5%), and I (2%), all of which are
descendents of the haplogroup N. Each haplogroup is associated with a different ancestral
lineage. (Because of its small size, sometimes haplogroup I is not considered to be a
major European group.) (Fitzpatrick & Yeiser, 2005).
DISCUSSION
Haplogroup I
About 2.2% of Europeans are descendants of mtDNA haplogroup I. Haplogroup I is one
of the oldest groups to inhabit Western Europe arriving there during the Upper Paleolithic
period –-about 30,000 to 40, 000 years ago -- and today is referred to as the Aurignacian
culture; most others arrived in Europe tens of thousands of years later as the Ice Age was
1
Extensive testing was also done relating to the author’s Y chromosome – referred to herein as nDNA –
but the length of a paper discussing both the mtDNA and the nDNA would be far in excess of what was
assigned.
2
These numbers vary by source.

3. in retreat.3
The Aurignacians were the earliest known Cro-Magnon culture. This culture
was marked by certain tools and a pronounced artistic tradition. Aurignacian culture was
preceeeded by Mousterian culture, was contemporary with Perigordian culture, and was
succeeded by the Solutrean culture.
The Aurignacian culture was marked by a great diversification and specialization of
tools, including the invention of the “burin” – an engraving tool -- that made much of
the art possible. The Aurignacian differs from other Upper Paleolithic cultures mainly in
a preponderance of stone flake tools rather than blades. Flakes were retouched to make
nosed scrapers, ridged scrapers, and end scrapers. Bones and antlers were made into
points and awls by splitting, sawing, and smoothing.
Aurignacian art is said to represent the first complete tradition in the history of art. Cave
art was produced almost exclusively in Western Europe, where, by the end of the
Aurignacian Period, hundreds of paintings, engravings, and reliefs had been executed on
the walls, the ceilings, and sometimes the floors of limestone caves. Probably the first
paintings were stencilings outlined in color of actual hands held against the cave walls.
The stencilings were followed by the development of figural painting. A characteristic
feature of these early pictures, which persisted throughout the Aurignacian period, is their
“twisted perspective,” which shows, for example, the head of the animal in profile and its
horns twisted to a front view. Classic examples of Aurignacian art are the paintings of
animals, such as horses and bulls, on the walls and ceilings of the cave at Lascaux in
southwestern France. These figures were painted in vivid polychrome red, yellow,
brown, and black, with solid and closed outlines.
The earliest examples of small, portable art objects produced during this period consist of
pebbles with very simple engravings of animal forms. Subsequently, animal figures were
carved in pieces of bone and ivory. In the later part of the Aurignacian Period the
carvings show increased naturalism with foreshortening and shading with cross-hatched
lines. (Aurignacian culture. (2008). In Encyclopædia Britannica. Retrieved July 23, 2008,
3
During this time, approximately 70,000 BC to 10,000 BCE, a mile-deep ice sheet covered the northern
part of the continent from just north of London in England and west to the mainland covering all of
Scandinavia and Poland. The glaciers in the Alps would have grown tremendously as well, fanning out in
all directions to provide an ice cap covering the top of the entire boot of Italy. Sea levels were
approximately 130 yards lower than today, meaning some Mediterranean islands (as well as some Atlantic
islands such as the Azores and the Canaries) would have much greater landmass than today, and even
islands such as Malta were connected to the mainland, just as Britain and Ireland would have been.
Ice Age Euroasia was cold, with temperatures on average 8 to 12 degrees Centigrade cooler than today, and
much drier. The European treeline ran from the Bay of Biscay in Spain, across the very north of Italy and
over to the Sea of Azov, where it dipped southeast toward the border between Iran and Turkmenistan. In
between the tree line and the glacial line, the landscape would have been dry, covered with sand dunes and
tough, wiry grass, perhaps similar to some extent to terrain associated with Iceland and Northern Canada
today.
This grassy intermediate zone -- known as the Great Eurasian Plain -- would have served as a natural game
corridor, running all the way from eastern France to Korea. Great herds of game roamed this grassy
thoroughfare: wooly mammoths, antelopes, and, in particular, a number of species of bison.

4. from Encyclopædia Britannica Online:
http://www.britannica.com/EBchecked/topic/43368/Aurignacian-culture; and
Time and Space The Archaeological Context. Retrieved July 23, 2008 from:
http://www.culture.gouv.fr/culture/arcnat/chauvet/en/espa2.htm).
Mitochondrial DNA
Organizational Display of Regions of MtDNA.
It is speculated that mitochondria were originally bacteria and were “engulfed” by and
developed a symbiotic relationship with other life forms living in their cellular

5. cytoplasm. Mitochondria provide their hosts with ATP in exchange for protection, etc.
Significantly, mitochondria have their own DNA i.e., their DNA is distinct from the
DNA (henceforth as nDNA for nuclear DNA) of their hosts. Mutations in mtDNA are
distinct from changes to the nDNA. 4
(Hart, 2002).
MtDNA mutates at a very slow rate -- over tens of generations -- and thus can be used to
glimpse the past.
MtDNA is understood to be inherited only from the mother. At conception, essentially
only the male sperm’s nucleus enters the female egg. In the case wherein male mtDNA
enters a female egg, human eggs are estimated to have as many as 100,000 mtDNA
molecules which would result in an extreme dilution. Yet more, the embryo’s cellular
machinery is able to identify any male mtDNA that has entered the egg and destroy it.
Thus, mothers pass along their mtDNA to their children and share similar mtDNA with
their siblings and maternal relatives and, as such, barring a contemporaneous mutation,
an individual’s mtDNA is not unique to him or her.5
(Gibson & Muse 2002).
When remains are old or badly degraded it is often difficult or impossible to extract
nDNA . However, mtDNA is present in much higher numbers than nDNA and so some
of it is more likely to remain testable. In short, while nDNA contains much more
information, there are only two copies (one paternal, one maternal) of it in each cell,
while mtDNA has a smaller amount of useful information but typically hundreds of
copies of that information in each cell; the use of mtDNA in this context simply reflects
its increased odds of not being destroyed.6
,7
(Goodwin & Linacre & Hadi, 2007).
MtDNA was first sequenced in 1981 from a European placenta which effort is referred to
as either the “Cambridge Reference Sequence” or as the “Anderson Sequence.” 8
4
MtDNA uses a slightly different code than nDNA. As examples:
(i) UGA in mtDNA codes for trytophan but is a stop codon in nDNA;
(ii) AUA in mtDNA codes for methionine whereas in nDNA it codes for isoleucine;
(iii) AGA and AGG both code for stops on mtDNA but code for arginine in nDNA.
(Goodwin, et al., 2007).
5
MtDNA has been one of the tools used (i) to identify the Vietnam Unknown Soldier, (ii) as part of the
process of identifying the Romanov family, (iii) to establish that Neanderthals were not direct ancestors of
modern humans, (iv) to discredit the claim of Anna Anderson Manahan to be the Russian Princess
Anastasia, and (v) to establish that Jesse James was indeed the person buried in his tomb. (Wells, 2003).
6
Both mtDNA’s circular structure and the encapsulation behind two walls further enhances the
survivability of mtDNA. (Gibson & Muse, 2002).
7
Interestingly, a portion of at least some humans’ chromosome 11 carries a portion of an mtDNA “control
region” reflecting an ancient transposition between mtDNA and nDNA. (Goodwin, et al., 2007).
8
Today the processes used 1981 – including the use of bovine sequences to fill in gaps – are referred to as
“rudimentary.” In 1999 the same placenta was resequenced with only 11 changes made to the 1981
standard (plus an additional 7 nucleotide positions actually related to rare polymorphisms), but most

6. On average, there are 4-5 copies of mtDNA per mitochondrion with a range of about 1 –
15 copies. Each cell typically contains hundreds of mitochondrion, with an estimated
average of about 500 per cell. (Gibson & Muse, 2002).
MtDNA exists as a circular structure. Typically, mtDNA has 16,569 9
base pairs (some
people have mutations).
MtDNA base pairs are counted clockwise.
Of these, 1,122 base pairs contain the origin of replication but do not control for any
other gene products. 15,447 of the 16,569 coding base pairs
importantly the two areas of primary focus – the HV1 and HV2 regions – were not adjusted. Actually, the
resequencing established there were only 16,568 base pairs but the numbering system keyed to 16,569 base
pairs was retained for the sake of reference consistency. However, it must be noted that some institutions
use different reference-systems -- one example is based on an African who had 16,571 base pairs -- so it is
necessary to be aware of the reference-system being used. (Goodwin, et al., 2007).
9
By way of contrast, human nDNA consists of somewhere between 49,530,000 to 247,200,000 bases.
(Wells, 2007.)

7. Relative size of the D-Loop (HV1 & HV2) vis-à-vis the Coding Region.
code for proteins; there are no introns and only a few noncoding base pairs. (Gibson &
Muse, 2002).
MtDNA has 37 genes that code for the products used in the oxidative phosphorylation
process, a.k.a. cellular energy production; 13 of these code for proteins, 22 for transfer
RNAs (tRNA) and 2 for ribosomal RNAs (rRNA). (Gibson & Muse, 2002).
The two mtDNA strands are designated as the “H” (heavy) and “L” (light) strands. The
H strand has a greater number of guanine nucleotides (the heaviest of the four different
nucleotides) than does the L strand. Replication begins in a non-coding region of the H
strand (the displacement loop/D-loop). 10
10
The D-Loop (HV1 & HV2) is considered a non-vital part of the mtDNA because it does not have an
immediately useful biological function. Thus, whenever a mutation occurs in this region the individual
does not die from the mutation and might survive and pass the mutation along to the next generation.
(Ridley, 2000).

8. Close up of the Hypervariable Regions.
The H strand encodes for 28 gene products and the L strand transcribes 8 tRNAs and an
enzyme known as “ND6.” (Gibson & Muse, 2002).
Because the D-loop region does not code for any functional cell processes, there are
significantly more polymorphisms between individuals in this region. Due to the larger
number of polymorphisms in this region, lineage tracing typically focuses on two coding
regions in the D-loop known as HV1 11
and HV2 (H is for “hypervariable”). (Gibson &
Muse, 2002).
MtDNA has fewer repair mechanisms than nDNA and, not surprisingly, mtDNA has
higher rates of mutation than does nDNA. Even more, mtDNA lacks proof-reading
mechanisms increasing the mutation rate vis-à-vis nDNA during replication. Hence, it is
estimated that mtDNA’s mutation rate is 10 (some sources say 6 to 17) times higher than
nDNA’s which has the effect of making the tracing of descent via mtDNA that much
easier (of course, there are many advantages and disadvantages to each of mtDNA and
nDNA for these purposes). (Gibson & Muse, 2002).
At first glance, complicating matters is the fact that because of the relatively high rate of
mtDNA mutation an individual might – probably does – have more than one mtDNA
type, i.e., with different mtDNA sequences; these different “types” can be located in
different cells, the same cell, and even in the same mitochondrion! However, often these
11
Sometimes as HVR1 & HVR2; HVS1 & HVS2; or HVI & HVII.

9. mutations are found in very low numbers; it is rare to find more than one position so
mutated out of the nucleotides sequenced at HV1 and HV2 and, even more, “hotspots”
for such mutations at HV1 and HV2 have been identified. (Goodwin, et al., 2007).
A standard mtDNA analysis involves polymerase chain reaction (PCR) amplification of
nine overlapping fragments followed by sequence analysis/digestion of 12-14 restriction
enzymes in the HV1 and HV2 regions. The results are then compared to the Cambridge
Reference Sequence. The estimate is that the sequences in these two areas vary by 1 to 2
percent between unrelated individuals; typically, these mutations are found in two
particular “hotspots” in the two HV regions. (Goodwin, et al., 2007).
A weakness with the use of mtDNA is that some haplogroups 12
are common within some
populations. An example is an FBI study of 1655 Caucasians of whom 168 (a little over
10%) would not be “excluded” as possibly related! Hence, increasingly, SNP Marker
Haplogroup Backbone Tests are being used. It should be noted that to sequence all
16,569 base pairs provides a 12-fold increase in the ability to differentiate such otherwise
common haplogroups. (Goodwin, et al., 2007).
Even given all of the above, it must be remembered that the further one goes back in any
such tracings, ever more extrapolations (educated guesses) of expected rates of mutations
are used to “connect the dots.”

10. HV1 Test
The HV1 Test is the most informative mtDNA test 13
as:
1. The HV1 region contains an abundance of markers.
2. The HV1 region is easy to test. All 520 nucleotides in the entire HV1 region can be
read from a single test.
3. The HV1 region is well studied. Thus, there is more scientific data available for
markers in the HV1 region than any other region of the mtDNA.
All of the other test types serve to supplement the results of the HVR1 test. (Smolenyak
Smolenyak & Turner, 2004).
***** ***** ***** ***** ***** ***** ***** ***** ***** ****
This mutation is documented as follows:
* Location: 40
* Nucleotide Change: T>G.
***** ***** ***** ***** ***** ***** ***** ***** ***** ****
13
The HV1 test uses “DNA sequencing” technology to read all of the nucleotides from locations 16,001 to
16,520 (the entire HV1 region).

11. HV1 Sequence of Louis Sheehan (Location 16001 to 16520) compared to
the CRS:
16001
ATTCTAATTT AAACTATTCT CTGTTCTTTC ATGGGGAAGC AGATTTGGGT
16051
ACCACCCAAG TATTGACTCA CCCgTCAACA ACCGCTATGT ATyTCGTACA
16101
TTACTGCCAG CCACCATGAA TATTGTACaG TACCATAAAT ACTTaACCAC
16151
CTGTAGTACA TAAAAACCCA ATCCACATCA AAACCCCCTC CCCATGCTTA
16201
CAAGCAAGTA CAGCAATCAA CCtTCAACTA TCACACATCA ACTGCAACTC
16251
CAAAGCCACC CCTCACCCAC TAGGATACCA ACAAACCTAC CCACCCTTAA
16301
CAGTACATAG TACATAAAGC CATTTACCGT ACATAGCACA TTACAGTCAA
16351
ATCCCTTCTt GTCCCCATGG ATGACCCCCC TCAGATAGGG aTCCCTTGAC
16401
CACCATCCTC CGTGAAATCA ATATCCCGCA CAAGAGTGCT ACTCTCCTCG
16451
CTCCGGGCCC ATAACACTTG GGGGTAGCTA AAGTGAACTG TATCCGACAT
16501
CTGGTTCCTA CTTCAGGGcC
Location Mutation Type 14
Nucleotide
16074 HV1 Substitution A > g
16093 HV1 Substitution T > y
16129 HV1 Substitution G > a
16145 HV1 Substitution G > a
16223 HV1 Substitution C > t
16360 HV1 Substitution C > t
16391 HV1 Substitution G > a
16519 HV1 Substitution T > c
A total of 8 mutations.
14
Substitution - occurs with a nucleotide changes.
Deletion - occurs when a nucleotide is removed from the sequence, therefore changing the overall
sequence.
Insertion - occurs when a nucleotide is added to the sequence, therefore changing the overall sequence.

12. HV2 Test
The HV2 region spans positions 1 to 400. Like HV1, HV2 contains an abundance of
markers that are useful for tracing maternal ancestry.
The HV2 test 15
supplements the HV1 results as it allows more stringent and precise
results for any comparison. For example, in cases where people are matching exactly at
the HV1 region, further comparison of the HV2 region provides greater resolution.
(Rodden Robinson, 2005).
HV2 Sequence of Louis Sheehan (Location 1 to 400) compared to the
CRS:
1
GATCACAGGT CTATCACCCT ATTAACCACT CACGGGAGCT CTCCATGCAT
51
TTGGTATTTT CGTCTGGGGG GTgTGCACGC GATAGCATTG CGAGACGCTG
101
GAGCCGGAGC ACCCTATGTC GCAGTATCTG TCTTTGATTC CTGCCTCATC
151
CcATTATTTA TCGCACCTAC GTTCAATATT ACAGGCGAAC ATACTTACcA
201
AAGcGTaTTA ATTAATTAAT GCTTGTAGGA CATAATAATA ACAATTGAAc
251
GTCTGCACAG CCgCTTTCCA CACAGACATC ATAACAAAAA ATTTCCACCA
301
AACCCCCCNc CCCCCNCTTN TGGNCNCANC ACTTAAANNC ATNTNTGCCA
351
AACCCCAAAA ANAAANAANC CTAANACCNG CCTAACCANA TTTNAAATTT
Location Mutation Type Nucleotide
73 HV2 Substitution A > g
152 HV2 Substitution T > c
199 HV2 Substitution T > c
204 HV2 Substitution T > c
207 HV2 Substitution G > a
250 HV2 Substitution T > c
263 HV2 Substitution A > g
310 HV2 Substitution T > c
A total of 8 mutations.
15
The HV2 test also uses “DNA sequencing” technology. This test reads all of the nucleotides from
locations 1 to 400 (the entire region) of the mtDNA.

13. Single Nucleotide Polymorphism Marker (“SNP”) Haplogroup
Backbone Test 16
, 17
, 18
, 19
16
There is also a SNP Subclade Test which examines a special panel of markers in the Coding Region of
the mtDNA allowing determination of one’s “sub-clade.” However, at the moment, SNP Subclade tests are
available only for the following mtDNA Haplogroups: R, M, H.
17
The 20 markers that are included in this test and the haplogroups they define are:
SNP Location Mutations Haplogroups
2352 T > C L1b, L3e, U6b1
3594 C > T L0, L1, L2, L5
3693 G > A L1b, L2d
4312 C > T L0
4580 G > A V
4833 A > G G
5178 C > A
C > T D
7028 C > T H
7055 A > C
A > G L1
7598 G > A E
8618 T > C L3d
10086 A > G L3b
10310 G > A F
10400 C > T C, D, E, G, M, Q, Z
10873 T > C C, D, E, G, L, M, Q, Z
11251 A > G JT, J, T
11719 G > A Pre-HV, HV
12308 A > G K, U
12705 C > T B, F, H, J, K, P, T, R, U, V
14766 C > T HV
18
As each sequencing reaction can only test approximately 500 nucleotides at a time, many reactions
would be required in order to sequence (“DNA Sequencing”) the entire Coding Region, thus making it
costly. Also, there are very few mutations in the Coding Region, making it unnecessary to sequence every
single nucleotide in the Coding Region. As such, a process other than “DNA Sequencing” is used wherein
20 specific Coding Region markers are tested.
19
The Coding Region of the mtDNA is considered essential for the survival of the individual, so typically
whenever a mutation occurs in this region it is often lethal and the individual dies. Thus, mutations which

14. While the markers in the HV1 and HV2 Regions can be easily detected by sequencing,
the Coding Region is large, making sequencing (currently) impractical. The SNP
Haplogroup Backbone Test is a panel of 20 markers in the Coding Region which are
specific for haplogroup determination. (Fitzpatrick, 2005).
occur in the Coding Region are usually not passed down to future generations. For this reason, over a
period of thousands of years, many mutations accumulate in the D-Loop (HV1 & HV2), but few are found
in the Coding Region. (Olson, 2002).

15. SNP Backbone Test of Louis Sheehan:
SNP Location Mutations 20
SNP Identity Mutation
2352 T > C
T Negative
3594 C > T
C Negative
3693 G > A
G Negative
4312 C > T
C Negative
4580 G > A
G Negative
4833 A > G
A Negative
5178 C > A
C > T
C Negative
7028 C > T
T Positive
7055 A > C
A > G
A Negative
7598 G > A
G Negative
8618 T > C
T Negative
10086 A > G
A Negative
10310 G > A
G Negative
10400 C > T
C Negative
10873 T > C
T Negative
11251 A > G
A Negative
11719 G > A
A Positive
12308 A > G
A Negative
12705 C > T
T Positive
14766 C > T
T Positive
A total of 4 mutations.
20
The presence of a mutation is as informative as is the absence of a mutation. For example, mtDNA
haplogroup H is the most common haplogroup among Europeans, and is confirmed in part by the absence
of all SNP markers tested in the mtDNA SNP Backbone test. (Nicholl, 2002).

16. CONCLUSION
Maternal Markers
mtDNA HV1: 8 Mutations
mtDNA HV2: 8 Mutations
mtDNA SNP: 4 Mutations
This specific pattern of mutations confirms Lou Sheehan’s mtDNA Haplogroup to be: I.

17. Geographic migration of Haplogroup I over time.

18. Works Cited
Aurignacian culture. (2008). In Encyclopædia Britannica. Retrieved July 23, 2008, from
Encyclopædia Britannica Online:
http://www.britannica.com/EBchecked/topic/43368/Aurignacian-culture
Fitzpatrick, C. & Yeiser, A. (2005). DNA & Genealogy. Fountain Valley:
Rice Book Press.
Fitzpatrick, C. (2005). Forensic Genealogy. Fountain Valley, California: Rice Book
Press.
Genetic Geneology (2008) http://www.genebase.com/index.php; and
http://www.genebase.com/index.php (maintained by Genetrack). Retrieved July
15, 2008 from: http://www.dnaancestryproject.com/
Gibson, G. & Muse, S.V. (2002). Primer of Genome Science. (1st ed.). Sunderland,
Massachusetts: Sinauer Associates, Inc.
Goodwin, W. & Linacre, A. & Hadi, S. (2007). An Introduction to Forensic Genetics.
West Sussex, England: John Wiley & Sons, Ltd.
Hart, A. (2002). How to Interpret Your DNA Test Results for Family History and
Ancestry: Scientists Speak Out on Geneaolgy Joining Genetics. Lincoln,
Nebraska: iUniverse.
Nicholl, D. (2002). An Introduction to Genetic Engineering. (2nd
ed.). New York:
Cambridge University Press.
Olson, S. (2002). Mapping Human History. New York: Mariner Books.

19. Ridley, M. (2000). Genome The Autobiography of a Species in 23 Chapters. New York:
HarperCollins.
Rodden Robinson, T. (2005). Genetics for Dummies. Hoboken, New Jersey: Wiley, Inc.
Smolenyak Smolenyak, M. & Turner, A. (2004). Trace Your Roots with DNA. Rodale.
Time and Space The Archaeological Context. Retrieved July 23, 2008 from:
http://www.culture.gouv.fr/culture/arcnat/chauvet/en/espa2.htm
Wells, S. (2007). Deep Ancestry Inside the Ancestry Genographic Project. Washington,
D.C.: National Geographic Society.
Wells, S. (2003). The Journey of Man a Genetic Odyssey. New York: Random House
Trade Paperback.