DNA Analysis and the Shroud of Turin:

Development of a Shroud CODIS

Kelly P. Kearse

Abstract
Since its development in the mid 1980s, DNA analysis has become a standard
procedure utilized by law enforcement and legal systems in the forensic examination
of human remains, and to help establish or exclude a connection to a crime scene.
The recent progression of gene amplification and enrichment strategies, together with
next generation sequencing techniques, have made the analysis of ancient and
degraded DNA samples much more feasible than previously imagined. Human DNA
has been isolated from the Shroud of Turin, although the results remain rather
limited and controversial. Indeed, it is unknown if such DNA truly originates from
blood cells present on the cloth or is the result of contamination from exogenous
sources. Here, the potential and limitations of modern molecular biology techniques
in the analysis of the Shroud of Turin are reviewed, including the evaluation of both
nuclear and mitochondrial DNA.

Organization of the human genome: DNA structure and function

Deoxyribonucleic acid, or DNA, encodes the information important for the
construction of all living things, from simple bacteria to human beings (1,2).
Structurally, DNA consists of four bases, or nucleotides, which are symbolized by the
letters A,T,C,G. These four bases are the building blocks of DNA. Similar to varying
the arrangement of letters of an alphabet to create different words, which are then
joined together to form sentences, variation in the number and sequence of the four
bases (A,T,C,G) determines the identity and function of specific DNA regions. For
example, the DNA sequence TTCGGCCAT would encode a different set of instructions
than the sequence CTAGTGTCC or the sequence ATCCTTGCG, and so forth.
Essentially two types of DNA sequences exist: (i) genes, which encode specific
products necessary for certain cellular functions; and (ii) noncoding segments, that
are localized between genes and do not encode specific products. Together, genes
and noncoding segments represent a set of instructions that is necessary for the
creation and function of a particular organism. This genetic blueprint is referred to
as a genome.
Sequencing of the entire human genome was completed in 2003, and showed that
human DNA contains only 20,000-30,000 genes, a much smaller number than was
originally estimated (3,4). Genes account for only about 2% of total human DNA,
with the remainder being noncoding sequences. The purpose of noncoding DNA
remains to be determined; certain noncoding sequences may function to regulate the
expression of specific genes. Other noncoding sequences may be nonessential, i.e.
“junk DNA” that serves no identifiable purpose (2-4).
Within a cell, both genes and noncoding sequences exist together on DNA strands
that are wound around each other in the form of a double helix (Figure 1). Such
strands are said to be complementary because the “A” base always pairs with the “T”

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Figure 1. Organization of DNA within the cell. Coding segments (genes) and
noncoding segments exist on complementary strands of DNA. Complementary strands
twist together to form double helices, which, in turn, are supercoiled together into
chromosomes, localized in the nucleus (see text for details).

base on the opposite strand. Similarly, “G” and “C” bases are always paired together.
Thus, if one knows the sequence of one strand, the order of the complementary
strand may be inferred. Helices are supercoiled around each other, similar to wires
being twisted together, and packaged into chromosomes (Figure 1). Twenty three
unique pairs of chromosomes exist in the nucleus of all cells in the body, except for
two cell types: sex cells (sperm or egg), which have only half of this amount (23
chromosomes total, but no pairs); and red blood cells, which lose their nucleus as
they mature and therefore are devoid of DNA (1,2,5).

DNA profiling and evaluation of specific DNA regions of interest

The vast majority of DNA (99.9%) is the same in all human beings, with only one-
tenth of 1% of total DNA being unique for each individual (with the exception of
identical twins). However, because of the vast size of the human genome
(approximately 3 billion base pairs in length), such a small difference is sufficient to
distinguish with a high level of confidence if the DNA in question originates from the
same person or a different individual (1,2). Elucidation of the DNA sequence of a

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single region, in of itself, is not that discriminatory; however, when many such
regions are examined together, such analysis can establish the identity and
relationship of DNA samples with a relatively high power of discrimination. This is
the basis of DNA testing, or DNA profiling (1,2,6).
Standard (nuclear) DNA profiling involves the examination of highly variable
regions found among the 23 pairs of chromosomes that are known as short tandem
repeats, or STRs. STRs are short nucleotide sequences (~4 base pairs in length) that
are repeated numerous times in a row in noncoding DNA regions scattered
throughout the genome. A core of 13 STRs, plus the amelogenin gene to distinguish X
and Y chromosomes, is currently utilized in the combined DNA index system
(CODIS), a national DNA database that is kept on file by the Federal Bureau of
Investigation (FBI), (6-8). By comparing a battery of specific STR sequences between
one DNA sample and another, and considering their frequency among various
populations, their relatedness can be determined with a relatively high probability.
The greater the number of STR sequences that are examined, the higher the
discriminatory power in determining the relationship of the samples in question.
STR CODIS analysis is one of the most recognized standards by which an individual
may be excluded or included in a particular case of interest.
Sequences other than STRs may also be evaluated to determine which form(s) of a
particular gene is (are) present, for example, the ABO blood group genes (blood
typing), and the human leukocyte antigens (tissue typing). Numerous companies
offer genetic analysis services to the public (for a fee) to determine ancestry
relationships among family members; such DNA tests as similar in principle, but are
outside of the typical STR/CODIS analysis (6-8).

Mitochondrial DNA versus nuclear DNA

Typically, when DNA analysis is discussed in the media, it is nuclear DNA which is
being referenced as this constitutes the bulk of the human genome. However, in
certain cases additional DNA sequences may be evaluated that are located outside of
the nucleus in a cellular organelle termed the mitochondrion (Figure 2).
Mitochondria perform numerous cellular functions, most notably the generation of
cellular energy. The mitochondrial genome is much smaller than that of the nuclear
genome, containing only 37 genes total, and unlike nuclear DNA, which is wound
into chromosomes, mitochondrial DNA exists in the form of a small, circular closed
loop (Figure 2), (1, 9-13). Mitochondrial DNA is composed of the same building
blocks (the bases A,T,C,G) that are used to create nuclear DNA. While only one
nucleus exists within a cell, hundreds to thousands of mitochondria may be present,
each of which can contain several copies of mitochondrial DNA. Thus, unlike nuclear
DNA, which exists as only 1 set (2 copies) per cell, 100-10,000 copies of
mitochondrial DNA may be present (Figure 2), (1, 9-12). This property makes
mitochondrial DNA analysis particularly useful in situations where sufficient nuclear
DNA may not be available for evaluation, for example in aged samples where DNA
degradation is a concern (9-12).

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Figure 2. Nuclear DNA and Mitochondrial DNA. Nuclear DNA exists in the form of
23 pairs of chromosomes localized in a (singular) nucleus within a cell. One cell may
contain hundreds to thousands of mitochondria, each of which may contain several
copies of mitochondrial DNA, present in the form of a closed, circular loop. Together,
nuclear DNA and mitochondrial DNA comprise the human genome (see text for details).

Another major difference that exists between nuclear DNA and mitochondrial DNA
is their mode of inheritance. Unlike nuclear DNA, which is received from both the
mother and the father, mitochondrial DNA is inherited strictly from the mother.
During fertilization, only nuclear DNA from the sperm is effectively transferred to the
egg cell. The nucleus in a sperm cell is located in the head region, which binds and
penetrates the egg cell surface; mitochondria are located further down in the
midpiece section, which transfer into the egg cell, but are subsequently destroyed
(13,14). Thus, all surviving mitochondrial DNA in the fertilized egg cell and
developing embryo are of maternal origin. Both male and female offspring born of the
same mother carry identical mitochondrial DNA, which is the same as that of the
mother, and the mother’s maternal relatives (1,10,11). Males carry such
mitochondrial DNA sequences but do not transmit them to subsequent offspring
(Figure 3). Because mitochondrial DNA has a relatively high mutation rate,
mitochondrial sequences typically differ from one unrelated person to another. Thus,
it can be said with a relatively high confidence that no two individuals will express
identical mitochondrial DNA sequences unless they share the same mother,

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grandmother, or great grandmother, and so forth. Depending on the extent of
similarity present within specific mitochondrial DNA segments,

Figure 3. Maternal inheritance of mitochondrial DNA. In this example, the

parents give birth to two daughters (D1, D2) and two sons (S1,S2), all of which inherit
the mother’s mitochondrial DNA, as indicated by the purple shading. When the sons
and daughters marry, only the daughters (D1, D2) will pass their mother’s
mitochondrial DNA on to their offspring (G1-G4). The offspring of the two sons, (G5-G8),
will receive mitochondrial DNA from their respective mothers, i.e. the two women that
each son married (see text for details).

ancestry may be traced back to previous generations that existed hundreds of years
ago or before (1, 10-12). Likewise, it can also be determined with a relatively high
certainty that if two samples do not contain identical mitochondrial DNA sequences,
they are not maternally related. Therefore, it is extremely unlikely that two unrelated
people will share the same mitochondrial DNA. This property is especially useful
when determining the relatedness of different DNA samples to one another.
Similar to nuclear DNA analysis, the discriminatory power of mitochondrial DNA
analysis is proportional to the number of specific regions that are examined. Unlike
nuclear DNA analysis, however, mitochondrial DNA testing cannot distinguish
between a brother and a sister or even certain cousins that share the same maternal
lineage (see Figure 3).
Analysis of mitochondrial DNA is based on a similar principle as nuclear DNA
profiling, involving the examination of certain regions within the mitochondrial
genome. Specifically, sequences located within so-called hypervariable regions,

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termed HV1 and HV2, are the most studied. Additional regions within the
mitochondrial genome may also be included for more definitive analysis (1,10-12). As
mentioned above, due to the relative high copy number of mitochondrial DNA within
the cell, such analysis is particularly useful when limited amounts of DNA may be
present (10-12).

Previous DNA studies on the Shroud of Turin

DNA analysis of the Shroud has been reported; the most recent of these being over
15 years ago, when the sensitivity and capability of molecular biology techniques was
far less than that which currently exists. In the late 1990s, Garza-Valdes reported in
the book “The DNA of God” the sequencing of portions of three genes from threads
taken from Shroud bloodstains: the beta-globin gene (a subunit of hemoglobin), and
the amelogenin X and amelogenin Y genes, present on X and Y chromosomes,
respectively. The threads examined were from the left-hand area and the occipital
region (the back of the head). Garza-Valdes concluded “all three segments of human
genes tested were positive, indicating the blood of the Man on the Shroud came from
a human male” (15).
In 1995, Canale and coworkers performed DNA analysis of samples taken from
both the Turin Shroud (from the soles of the left and right feet) and the Sudarium of
Oviedo. Several sequences were examined, including the amelogenin X and Y genes,
THO1 (tyrosine hydroxylase), vWA (von Willebrand factor), FES/FPS (tyrosine kinase),
and F13A1 (coagulation factor XIII), (16). Two of these regions, THO1 and vWA, are
part of the CODIS standard 13 STRs (6,7). The authors reported that contamination
between male and female DNA, and all of the other sequences examined was evident
(16, 17). It was stated that the presence of male DNA on the Shroud was more
noticeable that female DNA, and contamination by persons who touched the cloths
was considered as a contributing factor (16,17). Consequently, such results were
considered essentially null and void. Contamination among DNA samples becomes
evident when the presence of additional, distinct forms of a gene (termed alleles) is
detected (1,2,6,8). While numerous forms of gene may exist within population (from
several to hundreds, to even thousands depending on the particular gene), a single
individual can express at most two forms: one inherited from the mother and one
from the father (see Figure 4). Thus, when greater than two forms of a gene (alleles)
are present, it can be concluded that the sample is composed of DNA from more than
one individual (Figure 4). Amplification of mitochondrial DNA, but not nuclear DNA,
has been reported from the Sudarium (18), although no sequence information was
made available. As far as the author is aware, evaluation of mitochondrial DNA from
the Shroud has not been reported.
In hindsight, there are two main concerns associated with the previous DNA
studies on the Shroud: first, no evidence exists to show that any of the reported DNA
sequences actually came from blood cells. All DNA sequences that have been
examined to date are shared between blood cells and other cell types in the body,
including skin cells (see below). Second, as previously suggested (16), any
confirmatory DNA analysis of the Shroud should include evaluation of multiple
sequences from numerous bloodstains to provide an accurate comparison of the

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reproducibility of these findings (see below). Such studies are important in
establishing that any DNA present originates from a single source. In previous

Figure 4. A generalized analysis of DNA samples. Here, the analysis of two

hypothetical genes (GENE A and GENE B) in two individuals is shown. A person may
express up to two forms (maximum) of a gene, one that is inherited from the mother and
one from the father. In this example, analysis of GENE A would not distinguish
between Individual #1 and Individual #2 as both express identical forms (A1 and A3).
In contrast, analysis of GENE B would distinguish between these two individuals as
Individual #1 and Individual #2 express multiple distinct forms (B4, B7 and B2, B9,
respectively). If a sample contains >2 forms of any gene (GENE B, in this example), this
is an indication that contamination has occurred, i.e. that DNA from more than one
individual is present (see text for details).

studies (16), it was suggested that further investigation carried out on a larger
number of samples taken from protected areas may make it possible to ascertain the
original traces of DNA present on the Shroud (and Sudarium). This is especially
relevant given the technological advances in DNA isolation and sequencing that have
occurred within the past two decades. Such rapid progress has resulted from a
combination of: (i) being able to amplify even minute quantities of DNA, over a billion
copies of a single DNA sequence can be generated in four hours time using the
polymerase chain reaction (PCR) method (19,20); and (ii) the development of
computers with the capacity to direct many automated functions in the sequencing
steps, and the ability to store and analyze large amounts of data (21). So-called next

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generation DNA sequencing methods have become so sensitive within the past few
years that well volumes in the zeptoliter (10-21 L) range, containing a single DNA
replication enzyme, may now be evaluated (21). To put this into perspective, such
analysis is able to effectively examine a volume as small as one millionth of one
billionth of a single drop. It is now feasible to obtain sequence data from a single
DNA molecule (even without amplification), something that was unprecedented as
recently as 10-15 years ago.
Finally, it is noteworthy to mention that all previous Shroud DNA studies were
performed prior to the development of more sensitive sequencing techniques, relying
primarily on PCR amplification for DNA detection. PCR is most efficient at copying
DNA fragments that are at least 80 bases in length, which could be problematic for
certain aged DNA that might exist in smaller pieces. Next generation sequencers are
able to read each base separately, which has made analysis of many ancient DNA
genomes (often isolated in small fragments), now possible (21-28). In addition,
modified DNA sequencing methods are currently available that aid in study of highly
fragmented and degraded DNA, based on analysis of mini-STRs combined with single
nucleotide polymorphisms (SNPs). Modern multiplex analysis allows the examination
of multiple sequences within a single sample, minimizing the amount of material that
is required for study (21-28).

Development of a Shroud CODIS: Potential and limitations of DNA studies

Development of a Shroud CODIS (combined DNA index system) would involve
evaluating the presence or absence of a defined set of DNA sequences isolated from
various regions of the Shroud, to establish a collective profile of the DNA that exists
on the cloth. First and foremost, it would be important to determine the extent of
DNA heterogeneity that may be present by evaluating samples taken from multiple
bloodstains, including those on the reverse side. Conceivably, such distally located
threads may be relatively more protected from contamination as compared to those
on the regularly exposed side. Analysis of adjacent non-bloodstained fibers could
also prove useful in evaluating the extent of DNA contamination, in general, that may
be present within the region. For serious study, it would be worth considering that a
DNA sample (simple cheek swab) be submitted by all available and willing persons
that come in contact with the Shroud. Such samples could prove extremely useful in
helping to index any background DNA sequences that may exist. Similar to the
approach used in Human Genome Project, confidentiality could be maintained to
ensure anonymity of results (3,4). Essentially, there are three possibilities regarding
the origin(s) of any DNA present in Shroud bloodstains: i) The DNA is exclusively
representative of endogenous DNA from white blood cells; ii) The DNA is exclusively
representative of contaminating exogenous DNA (skin, sweat, saliva, tears); or iii) The
DNA is a mixture of both endogenous and exogenous sequences. Development of a
Shroud CODIS would help to discriminate between these various possibilities and
place in context any previous sequence data that has been obtained.
One of the most fundamental issues that remains to be determined regarding DNA
and the Shroud is if there is any DNA present that can be connected exclusively to
blood cells (20). To date, none of the reported DNA sequence analysis addresses this

8
primary question. These experiments would require evaluation of immune receptor
gene rearrangement, which is unique to white blood cells (5,20). Contaminating DNA
from skin cells or any other source than blood cells would test negative in such a
system. Another fundamental issue that has yet to be firmly resolved is the sex
typing of the bloodstains. As previously mentioned, testing of the amelogenin gene is
a standard addition in the 13 STR multiplex evaluation procedure. Sampling from
several bloodstains, together with specific evaluation to the degree of heterogeneity
that may be present (including additional STRs located on X, Y chromosomes) could
decisively establish the male or female genotype of the bloodstains. Another area of
interest that DNA analysis could potentially advance is to determine if the Shroud
bloodstains originate from a single individual. These data might also be useful in
addressing the suggestion that certain bloodstains have been “touched up” with the
successive addition of blood throughout the Shroud’s history. Finally, through
probing of specific target sequences, the ABO and Rh factor genes, it might be
possible to confirm the shroud blood type genetically, which would extend previous
studies using serological techniques (29,30).
Taken together, such studies could establish the collective profile of the DNA
sequences that are present on the Shroud. By extension, this Shroud CODIS could
potentially be used to determine if a similar (identical) DNA profile was present on
related artifacts, for example the Sudarium of Oviedo or the Tunic of Argentuli.
The two main limitations that most likely exist in the DNA analysis of the Shroud
are: (i) the limited amounts of endogenous DNA that may be present and (ii) the
amount of heterogeneity (contamination) that may exist. The discriminatory power of
STR (and mitochondrial DNA) analysis is a function of the number of sequences for
which data is collected. Definitive results from all 13 STR loci, which may not be
achievable, would provide the highest discriminatory power to define the DNA profile
representative of a particular Shroud bloodstain. Comparison of multiple STRs
(and/or mitochondrial DNA) among bloodstains would provide an indication if DNA is
present from a single or multiple source(s). How many different types of sequences
might be required for a confident identification of the Shroud DNA profile is difficult
to estimate: exclusion based on disparity relies on much simpler statistical
probabilities than inclusion due to multiple matching (2,6,8).
It is unknown if degradation issues would preclude data collection and analysis
even with more modern sequencing methods that are now available. Previous reports
have indicated that DNA is, in fact, present on the Shroud, albeit in the pre-
restoration time period (15,16). The restoration procedures may have introduced
additional, exogenous DNA samples on the cloth, or perhaps even removed a degree
of superficial, contaminating DNA from certain sites. Contamination could be a major
issue; comparative evaluation of threads taken from multiple bloodstains should
provide some insight as to the degree of heterogeneity that may exist. Parallel
examination of sequencing data from a singular bloodstain sampled from both the
front and reverse sides of the cloth might prove particularly informative.
Finally, DNA studies are by nature comparative. However, it should not be
misunderstood that evaluation of DNA sequencing data would allow scientists to
determine the identity of the man on the Shroud. No historic DNA profile of specific
individuals, i.e. Jesus, exists for comparison. Provided sufficient DNA was intact, it

9
is conceivable that a more detailed evaluation of sequences (outside of standard
CODIS STRs) could potentially provide certain information related to ancestry. It
should be emphasized, however, that inclusion or exclusion to a particular heritage
or region is not based on the evaluation of single, or even several, gene(s), but
requires the analysis and consideration of multiple factors. At this stage, any
suggestion of putative expected results would be unjustified speculation. Indeed, it
remains to be determined if any endogenous DNA is even present and if standard
sequence analyses of multiple regions are tenable. The generation of a Shroud
CODIS would be most useful for internal comparison, among bloodstains, to
conclusively establish what the DNA profile of the bloodstains consists of. DNA
sequence data could provide insight into many important fundamental questions
about the nature of the bloodstains, which at present remain inconclusive.
Depending on the fidelity of such results, such data could possibly be extended to
other, related artifacts to support or exclude their potential relationship with each
other.

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Acknowledgements

Thank you to John LaForest and Barrie Schwortz for critical reading of the
manuscript. As always, thank you to my wife, Kathy, for everything.

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