Scheffler Immo E. Mitochondria

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HUMAN EVOLUTION 421

There are additional disputes about the calibration of the clock. Is the rate

of change the same at all sites where changes have been observed? Do non-

coding regions (D - loop) change more rapidly than coding regions? Are the

changes in the third positions of the codons occurring at a faster rate? What

about the relative rates of transitions versus transversions in a DNA sequence?

The challenge was taken up by Hasegawa et al. (17) to predict a more accurate

time scale for human mtDNA, making the additional assumption that the

divergence of humans from our closest primate ancestor, the chimpanzee, was

about 4 million years ago. The emergence of modern humans out of Africa

was moved to an even more recent date: 89,000 ± 69,000 years ago. Such a

result also put the authors squarely at odds with the multiregional hypothesis

of modern human origin, which has gained adherents in recent years. Uncer-

tainties and controversies related to the mutation rate in mitochondria have

by no means been resolved to everyone ’ s satisfaction, and as a result the esti-

mates for the age of the mitochondrial Eve range from < 100,000 years to

almost a million years (see below).

After the African mitochondrial Eve was declared “ wounded but not dead

yet ” (18) in 1992, based on the ambiguity surrounding the construction of

phylogenetic trees with mtDNA sequences, supporters of the hypothesis have

assembled independent support. The controversy has at times been heated.

An attempt by a respected expert to debunk the “ myth of Eve ” (19) was fol-

lowed a few issues later by a rebuttal by another expert accusing the ﬁ rst of

perpetuating another myth (20) . As Ayala states succinctly, the coalescence of

the mtDNA sequences from over 100 ethnically diverse individuals to one

ancestral sequence, that of the “ mitochondrial Eve, ” is a property of all gene

genealogies, but does not mean that all humans descended from a single

mother. While Wilson and his colleagues probably also did not mean to be

taken literally, the statement had an attention - catching appeal, and the popular

press made the most of it. If not a single mother, there is still the question

about the size of the pool of humans from which the present - day human

groups are descended. The current controversy therefore centers around two

or three problems. Where is the mtDNA genealogical tree rooted? Evidence

points strongly toward Africa, but it is not yet conclusive. The second question

is concerned with the existence of a bottleneck, as well as the size of the bottle-

neck of those individuals who are the ancestors of all modern humans. Finally,

there may have been multiple bottlenecks with subsequent mixing of the sur-

viving populations. The arguments about multiregional models require exten-

sive migrations and interbreeding between populations to account for a single

gene pool (19) .

Ingenious independent analyses have strengthened the out - of - Africa

hypothesis. To establish the true root of the phylogenetic tree, the true human

mtDNA ancestor, one must choose an outgroup sequence from a different

species more ancient than humans, but still closely related. The chimpanzee

mtDNA has served such a purpose; but in the control region used routinely,

the rate of change may be too fast and too extreme. The creation of multiple

422 MITOCHONDRIAL DNA SEQUENCING AND ANTHROPOLOGY

and parallel sequence changes can complicate and obscure the analysis. A

novel ﬁ nding was exploited elegantly by Zischler et al. (21) to identify a supe-

rior outgroup for rooting the human mtDNA tree. Following the original

discovery of Fukuda et al. (22) , several investigators have conﬁ rmed that the

human nuclear genome contains several hundred copies of sequence frag-

ments closely resembling mtDNA. Such pseudogenes have also been found in

other primates and rodents, and their distribution and sequence alterations

make them useful molecular fossils in the nucleus (23) . Zischler et al. (21)

speciﬁ cally searched for and found a nuclear copy of the human mitochondrial

D - loop which had been transferred to the nucleus after the divergence of

humans from the chimpanzee, but before the “ growth ” of the human mito-

chondrial phylogenetic tree. The nuclear D - loop sequence became a superior

out group for rooting the tree in Africa (24) .

The mtDNA data assembled by many groups — prominently among them

the group at Emory University, as well as Stoneking and colleagues at Penn-

sylvania State University — have generally supported the following scenario

for the expansion of Homo sapiens around the globe (25) : The migration

started out of Africa about 100,000 to 200,000 years ago. It split somewhere

in the middle east into a group headed for central and northern Europe

(∼ 50,000 years ago), and another group headed toward southern and southeast

Asia. Migrations from present day China via the Indonesian Archipelago

toward New Guinea and Australia also occurred a long time ago. Somewhat

later the populations expanded north along the eastern rim of Asia, and even-

tually groups reached the Bering Strait to cross into North America. There

may have been several crossings of the Bering Strait (see below). Expansion(s)

from Alaska to the south populated in order North America, Central America,

and South America. Quite recent in human history, another wave of expansion

occurred from the islands surrounding New Guinea and North Australia into

the northern Paciﬁ c (Polynesia and Hawaii).

Of course, many of these deductions were not completely new. Data from

paleoanthropology and archeology had already been used to make broadly

similar deductions. Linguistic comparisons have also contributed greatly. Nev-

ertheless, mtDNA sequence data can often provide detailed and convincing

support. For example, a 9 - bp deletion in the small intergenic region between

COXII and tRNA

Lys

is believed to have arisen in Asia about 50,000 years ago.

Redd et al. (26) have traced a speciﬁ c set of control region mutations ( “ the

Polynesian motif ” ) associated with the 9 - bp deletion to a Taiwan origin (about

6000 – 8000 years ago), and from there across the Philippines, Indonesia, along

the coast of New Guinea, and ﬁ nally into Polynesia (26) . These molecular data

are in excellent agreement with the postulate of a southeast Asian origin of

Polynesians based on archeological and linguistic evidence.

The contention about rooting the human phylogenetic tree may have

approached a consensus, or at least the African root has acquired the support

of the majority. It is yet another issue to date the period of the existence of

the mitochondrial Eve. Determining the age of Eve is important, because an

HUMAN EVOLUTION 423

accurate estimate of the time span to the present serves in arguments about

other theories, speciﬁ cally the multiple origins theory. Since the fossil record

reveals the existence of Homo erectus as long as at least a million years ago,

and such evidence is found in Europe, the Middle East, and Asia, one is forced

to conclude from the genetic evidence that the modern humans (our ances-

tors) displaced all of these other human populations completely without any

detectable genetic exchange. If the lowest estimate for the age of the mito-

chondrial Eve is accepted ( < 100,000 years), this scenario would be quite aston-

ishing. On the other hand, if the mitochondrial Eve lived more than 500,000

years ago, a new set of questions and theories can be entertained about mul-

tiple origins, shifts in the composition of the human gene pool, and selective

pressures that might have contributed to the unusual evolutionary pattern. A

thoughtful reexamination of existing data sets by Wills (27) comes to the con-

clusion that Eve may be between 436,000 and 806,000 years old, an estimate

that is also very dependent on the estimate of the time of the split between

the human and chimpanzee lineages.

In the past decade there has been an avalanche of sequence data for

mtDNA, the Y chromosome, and autosomes from a wide range of human

donors. An up - to - date analysis of the conclusions has appeared from Garrigan

and Hammer (28) . These authors clearly distinguish two epochs in the evolu-

tion of H. sapiens . The ﬁ rst, starting about 2 million years ago, leads to the

emergence of the “ anatomically modern human. ” The second epoch covers

the geographic distribution of the modern humans after their emergence from

Africa. Sequence analyses are used to construct gene trees, and these in turn

can be interpreted to estimate the “ time to the most recent ancestor ” (TMRCA),

with certain assumptions about the molecular clock for accumulating nucleo-

tide changes. The mtDNA tree from the complete sequences of a global sample

of humans yields a TMRCA of ∼ 170,000 years. From the tree based on the

nonrecombining regions of the Y chromosome the estimate for the TMRCA

is roughly half this value, and various explanations have been offered (see

reference 28 ). As more and more sequence data from loci on the X chromo-

some or autosomes become available, the TMRCA can be pushed back to

500,000 – 800,000 years and thus include H. neanderthalensis, H. heidelbergensis ,

and other now extinct lineages of H. sapiens . Some of these results may con-

tradict the “ single origin model ” derived from mtDNA and Y chromosome

trees, and the multiregional evolution model may gain favor in the future. The

interested reader is directed to this highly technical paper for a consideration

of alternate models. Some support for the claim that modern humans from

Africa actually had “ close physical contact ” (does that mean sex?) with archaic

humans ( H. erectus ) comes from an unexpected direction. A genetic analysis

of lice revealed that there are two subspecies, one thought to have lived on H.

erectus and the other on H. sapiens . After a relatively long period of isolation

of both the lice and the humans, physical contact between these two groups of

humans before H. erectus became extinct must have taken place to explain the

present - day distribution of these louse subspecies in the world (29, 30) .

424 MITOCHONDRIAL DNA SEQUENCING AND ANTHROPOLOGY

Mitochondrial DNA sequence analysis has been perfected to the level

where analyses have become possible with samples from a 4000 - year - old

mummy of an Egyptian priest and from a 7000 - year - old human brain from a

Florida peat bog; and if the bones are ever released for analysis before they

are re - buried, there are paleobiologists waiting to obtain samples from a skel-

eton found on the northwest Washington coast thought to be 9000 years old.

One of the most impressive achievements in the study of human evolution by

the study of human DNA sequences was reported by Krings et al. in 1997 (31) .

The group in Munich, with independent conﬁ rmation by a group in Pennsyl-

vania, reported mtDNA sequences from a Neanderthal person. The Neander-

thals were a race of humans roaming through Europe and Western Asia from

about 300,000 to 30,000 years ago. Considerable discussion had centered

around the question whether Homo neanderthalensis was a distinct and sepa-

rate species from Homo sapiens . Their range overlapped in Europe during the

more recent millenia, and the possibility of a mixing of the gene pools by

interbreeding had not been convincingly excluded. In the landmark paper

published by the Paabo and Stoneking groups the deﬁ nite conclusion was

reached that the Neanderthals went extinct, leaving no trace of their mtDNA

in modern humans. Concentrating on the D - loop region, sequence compari-

sons and phylogenetic analyses were made, including a large number of rep-

resentatives of modern humans as well as the chimpanzee. The divergence of

the Neanderthal from modern humans was estimated from the mtDNA analy-

ses to have occurred about 500,000 years ago, in satisfactory agreement with

other estimates based on the archeological record.

Krings et al. (31) is a landmark for its conclusions, but it probably also set

the standard for future studies of ancient DNA. The DNA was retrieved from

a fossilized bone of an individual living between 100,000 and 30,000 years ago.

As discussed in more detail in this paper and elsewhere, measurements of

amino acid racemization can give reliable estimates about preservation and

the feasibility of obtaining sufﬁ cient DNA from a fossil specimen, and the

Neanderthal bone may be near the limit of what constitutes a useful source.

The quantitative considerations also indicate that only mtDNA sequences can

be expected to be recovered for PCR - based ampliﬁ cation, because it is several

orders more abundant than nuclear DNA. In a commentary on this paper in

the same issue of Cell , T. Lindahl discusses the “ ﬁ asco of DNA from insects in

amber, ” with reference to studies suggesting that amber is a very poor material

for the preservation of DNA (but not of the insect exoskeleton) (32) . Thus,

claims of DNA ampliﬁ cation from million - year - old insects in amber may con-

tinue to stimulate Hollywood, but they cannot stand up to rigorous scrutiny.

Because such skepticism is clearly indicated, the Neanderthal mtDNA paper

went to extraordinary lengths to include controls and to obtain repeatable

results, even when conducted in a distant, second laboratory. Since this paper

appeared, more partial sequences from Neanderthal mtDNA have appeared,

conﬁ rming the original conclusions in favor of the “ rapid replacement ” model

HUMAN EVOLUTION 425

for the origin of modern humans and the absence of any interbreeding between

Neanderthals and modern humans (33, 34) .

Another, more limited application of mtDNA sequence analysis to human

evolution and history is also instructive, but ultimately bafﬂ ing to the outsider

because of still unresolved controversies. Anthropologists have been curious

for some time about the following questions: When were the Americas popu-

lated by humans, and where did these people originate? Before the advent of

molecular genetic analyses, several time - honored approaches had suggested

answers, which cannot be discussed here in detail. The more conventional

archeological studies use the various artifacts (pottery, tools, weapons) to trace

lineages based on designs, patterns, materials used, and so on. A more recent

development is the application of the tools of linguistics to the comparative

analysis of the languages spoken by the various native Americans (North and

South America). Such an analysis had suggested three distinct linguistic divi-

sions: Amerind (spoken by American Indians in North, Central, and South

America), Eskimo - Aleut (spoken by the Inuit (Eskimo) and Aleutian Island-

ers), and Na - Dene (spoken by the by people of the Northeast Coast of Canada

and the United States as well as the Navajo and Apache, believed to have

migrated south to their present location around 1000 bc ). An interpretation

was that there were three distinct waves of immigration across the Bering

Straits, in agreement with other archeological data, such as distinctive features

of molar shapes. Mitochondrial DNA data assembled by Wallace, Torrioni, and

colleagues (reviewed in reference 25 ) not only conﬁ rmed the Asian origin of

the native Americans, but appeared to lend strong support to the three waves

of migration hypothesized by the linguists. Four haplogroups were found based

on extensive restriction mapping, and the relative abundance of these groups

could be interpreted in terms of three distinct migrations separated in time by

thousands of years.

Somewhat ironically, an ex - student of Wallace has collected additional data

that challenge the interpretation of his mentor (35) . The new ﬁ ndings of all

four haplotypes among the three linguistic groups (mtDNA samples from 1300

American Indians) appear to ﬁ t better to a scenario in which a single wave of

immigration brought women with all four haplotypes across the Bering Strait.

Subsequent migrations, along with the possibility of severe temporary reduc-

tions in one or more local populations due to environmental inﬂ uences, may

have caused the loss of particular haplotypes (another bottleneck), resulting

in the current distributions.

The discussion continues. It is not the intent here to present the deﬁ nitive

conclusion, if there is one, but to make the reader appreciate the wealth and

diversity of ideas derived from this research.

There are now well in excess of 2100 mitochondrial DNA sequences in the

Human Mitochondrial Genome Database (mtDB) ( http://www.genpat.uu.se/

mtDB ), including more than 1544 complete sequences (36) . The database also

lists more than 3000 sequence polymorphisms from human population groups

426 MITOCHONDRIAL DNA SEQUENCING AND ANTHROPOLOGY

all over the globe. This is a treasure trove for human population geneticists

and medical researchers interested in correlations between mtDNA haplo-

types and phenotypes. The “ harvesting of the fruit from the human mtDNA

tree ” is in full swing (37) , with new disciplines, “ archeogenetics, ” “ phylogeo-

graphy, ” and so on, being created. Another exhaustive database is discussed

by Attimonelli et al. ( http://www.hmdb.uniba.it ) (38) .

Regional variation in mtDNA haplotypes has generally been attributed to

genetic drift and migration. The question has been raised as to whether some

of these sequence variations in coding regions might have functional signiﬁ -

cance (39) and whether a selective advantage could arise for a population in

a speciﬁ c environment (40) . The authors conclude that some mtDNA lineages

found in European, Asian, Siberian, and Native North Americans might reﬂ ect

adaptations to a cold climate. Such an advantage could be achieved by a slight

decrease in the coupling of electron transport and ATP synthesis in favor of

thermogenesis. However, at this time such a conclusion must remain quite

speculative.

8.3 PRIMATE EVOLUTION

Phylogenetic analysis of mitochondrial DNA sequences can of course be

pushed back in time to include other species. No signiﬁ cant surprises about

evolutionary history may be expected when comparing different vertebrates,

or even different mammals. However, for calibrating molecular clocks and for

establishing more precisely the time of divergence of the nonhuman apes, a

comparison of the mtDNA sequences of humans, chimpanzee, pigmy chim-

panzee, gorilla, and orangutan has been very informative. Horai et al. (41)

found these genomes to be highly homologous, with a synonymous substitu-

tion rate of 3.89 × 10

− 8

/site per year, if one assumes that the orangutan and

the African apes diverged 13 million years ago. In the D - loop region this rate

was almost twice as high. The analysis also included three human mtDNA

sequences from Africans, Europeans, and Japanese to shed light on the origin

on modern humans. From the estimated substitution rates the age of the last

common ancestor of modern humans was calculated to be 143,000 ± 18,000

years, and the hypothesis of the African origin of Homo sapiens sapiens

received additional support.

The most detailed examination of the noncoding control region (human

nucleotide positions 16053 – 16465) of the nine taxa of African or African -

derived hominoids (humans, chipanzees, bonobos and gorillas) has been

undertaken by Gagneux et al. (42) , who examined 1158 unique haplotypes

including 811 humans from around the world. A phylogenetic tree was con-

structed which also included orangutans and the newly established sequence

from Homo neanderthalensis (Figure 8.1 ). For details on the computer analysis

and related analytical methods, the reader is referred to the original paper.

The ﬁ nal tree shown was also subjected to various degrees of topiary pruning

Figure 8.1 Primate evolution based on the analysis of mtDNA. An unrooted phylo-

gram of the neighbor - joining tree of 1158 different mitochondrial control region

sequences was constructed (A) and then subjected to topiary pruning (B). See Gagneux

et al. (42) for details. [The ﬁ gure was generously provided by Drs. Gagneux and Wills

prior to publication (with permission).]

(16) , but only one intermediate level is shown here. The tree illustrates in

dramatic fashion some previous conclusions, and it adds some novel insights.

It is clearly supportive of the closer relationship between humans and chim-

panzees relative to the other great apes. Most remarkably, it reveals deep

branches between chimpanzees in various African locations, corresponding to

the existence of several subspecies, and a previously unrecognized subclade of

PRIMATE EVOLUTION 427

428 MITOCHONDRIAL DNA SEQUENCING AND ANTHROPOLOGY

chimpanzees in Nigeria and northern Cameroon. There is signiﬁ cantly more

variation between the major chimpanzee clades than between the entire

human population. The appearance of the tree and speciﬁ cally the relatively

shallow human branches are consistent with the idea of a demographic bottle-

neck in human evolution — in other words, consistent with the idea that the

present human lineage arose from a small effective population of relatively

recent origin. A mitochondrial DNA fragment that entered the nuclear genome

was also included: It originated after the divergence of humans from the

great apes, and even after the divergence of present humans from the

Neanderthals.

An interesting corollary to these studies was published by Kenyon and

Moraes (43) . These authors used human ρ

cells (mtDNA - less) in fusions with

enucleated cells from the hominoid apes chimpanzee, pigmy chimpanzee,

gorilla, and orangutan to produce “ xenomitochondrial cybrids. ” Cybrids with

chimpanzee or gorilla mtDNA and a human nucleus could respire and carry

out oxidative phosphorylation. In contrast, the orangutan mtDNA and the

human nuclear genome were no longer compatible. The results support the

notion that the nuclear and mitochondrial genomes have to co - evolve at a

signiﬁ cant number of loci in order to maintain the critical interactions between

OXPHOS proteins encoded by the nucleus and proteins encoded by the mito-

chondria. Thus, the more closely related ape mt genomes could be functionally

expressed to interact with human nuclear gene products, but the orangutan

mt genome had evolved far enough since the distant separation of the lineages

to yield mitochondrial proteins which presumably could no longer interact

with human proteins in the inner mitochondrial membrane. It will now be a

challenge to distinguish simple polymorphisms with no structural – biochemical

consequences from those nucleotide changes (and the resulting amino acid

substitutions) which contribute to the defective interactions between nuclear

and mitochondrial gene products. Unfortunately, the use of recombinant

(human – primate) mtDNA molecules is technically still impossible. On the

other hand, the available crystal structures for complexes III and IV may guide

the search for protein – protein interaction involving nuclear - coded and

mitochondrial peptides and may perhaps reveal critical contact sites.

8.4 HUMAN Y CHROMOSOME VARIATION

A crucial, inherent advantage of the mitochondrial genome in the analyses

described above is (a) the absence of recombination, facilitating the construc-

tion of phylogenies, and (b) the estimation of the time intervals elapsed

between the different branch points, assuming that the molecular clock is

running at a constant rate. Another chromosome that has long been recog-

nized not to participate in recombination is the Y chromosome. The statement

has to be qualiﬁ ed, since a portion at the tip of the short arm, the pseudoau-

tosomal region, does participate in chiasma formation and recombination. The

male - speciﬁ c portion of the Y chromosome, however, offers an alternative and

complementary system for the study of human evolution and migratory path-

ways, and data on polymorphisms on this chromosome have been collected

and interpreted extensively. It is self - evident that the Y chromosome is inher-

ited along the paternal lineage, since it is the male - speciﬁ c sex chromosome

containing the critical gene (SRY) encoding the testis determining factor (44) .

By comparison with mtDNA, the Y chromosome is huge, comprising approxi-

mately 60,000 kb (60 Mb) of DNA. The great bulk of Y chromosome DNA,

especially the long arm (Yq), is made up of constitutive heterochromatin

consisting of various families of repetitive DNA with no obvious genetic

information. It has taken some time to deﬁ ne unique loci and to establish

polymorphisms at these loci in human populations (45 – 50) .

A detailed discussion of human Y chromosome polymorphisms would go

beyond the scope of this monograph. On the other hand, it is worthwhile to

consider how far the deductions from the analysis of mtDNA have been con-

ﬁ rmed by similar studies with Y chromosome sequences, and what kinds of

unresolved discrepancies exist. Independent conﬁ rmations of the reconstruc-

tion of human evolution are of course a source of intellectual satisfaction, and

they strengthen the conclusions drawn. Discrepancies, on the other hand, raise

issues about methodology, interpretation, and limitations of computer analy-

ses, but in the long run they can lead to new insights and progress.

A comprehensive and recent description of the geographic distribution of

human Y chromosome variation can be found in the publication by Hammer

et al. (50) , which includes data from 1500 males from 60 population groups,

subdivided into 15 major groups based on geographic, linguistic, and historical

information. In agreement with other genetic analyses, and speciﬁ cally the

mtDNA data, the Y - chromosome haplotype distributions show that sub -

Saharan African populations are distinct from the rest of the world, and they

exhibit the highest within - group diversity. Globally, the variants found outside

of Africa represent subsets of the haplotypes found within Africa. Subsequent

results from a more extensive sequencing of unique regions of the human Y

chromosome also supported the idea that “ Adam was an African ” (51 – 53) .

Hammer et al. were careful to point out that while such a within - group and

among - group pattern also supports the hypothesis of the origin of modern

humans from an older African population — with migrations of subgroups

leading to the population of distal sites, along with more limited genetic diver-

sity elsewhere resulting from bottlenecks — it may nevertheless be an overly

simplistic view. In the absence of additional information, the genetic data are

equally consistent with multiple short - range and long - range migrations, genetic

drift due to founder effects, and small population sizes (near extinction).

Africa may well have been the origin of more than one migration originating

from different parts of Africa. Notably, the Y chromosome data also are con-

sistent with the relatively recent ( ∼ 100,000 years) origin of our common ances-

tors, but may be a consequence of a small effective population size throughout

the Late Middle Pleistocene, rather than a true deﬁ nition of the period of

HUMAN Y CHROMOSOME VARIATION 429

430 MITOCHONDRIAL DNA SEQUENCING AND ANTHROPOLOGY

origin of Homo sapiens . For additional theoretical as well as methodological

considerations the reader is referred to several publications from Hammer

and colleagues (28, 50) , as well as the numerous additional publications cited

in these papers.

8.5 FORENSIC APPLICATIONS

Under other monarchies the male line takes precedence of the female in tracing

genealogies, but here the opposite is the case — the female line takes the prece-

dence. Their reason for this is exceedingly simple, and I recommend it to the

aristocracy of Europe: They say it is easy to know who a man ’ s mother was, but,

etc., etc.

— Mark Twain, “ Letters from the Sandwich Islands, ” describing the

Hawaiian Monarchy

Mitochondrial DNA sequences also mutate rapidly enough to create distinc-

tions (polymorphisms) between members of the same ethnic group — that is,

close “ relatives ” on an evolutionary scale, though not relatives in the legal

sense. Applications in forensic investigations have been made, and two such

applications will be used as examples to illustrate the methodology and the

potential limitations.

Attention is focused on the mtDNA control region (D - loop). In 1983 in a

relatively small sample of Caucasians and Africans it was ﬁ rst found that there

was ∼ 1.7% nucleotide diversity in ∼ 900 basepairs, with most of the differences

clustered in two hypervariable regions (54) . The 347 - basepair hypervariable

region (within nucleotides 16023 – 16388) was later analyzed by Orrego and in

10 additional unrelated Caucasian individuals of European ancestry. In the

combined data set from Greenberg et al. (54) , Anderson et al. (55) , and Orrego

and King (55a) , 32 sites out of 347 sites compared were found to be variable,

and pairwise comparisons showed differences ranging from 1 to 13 nucleotides

(average 5.9). A formal statistical analysis revealed that the observed and

expected number of sequence differences between pairs of individuals closely

approximated a Poisson distribution, which allows one to calculate that the

probability of two unrelated Caucasian individuals having an identical

sequence of 347 basepairs is 1 in 370 (see reference 56 ) for a discussion of

these results and related issues).

With oligonucleotide primers corresponding to the conserved ﬂ anking

sequences (tRNA

Pro

and tRNA

Phe

), it is a routine matter to amplify the D - loop

region from very small tissue samples. As described by King (56) , a second

ampliﬁ cation with nested but unequal amounts of primers can yield a single -

strand product ready for DNA sequencing. Optimization of conditions to

reduce artifacts from polymerase errors is part of the standard methodology,

and mosaics created with unintended target sequences can be detected and

eliminated because the normal target is so well known. A successful test analysis