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DNA-analytical tests on fresh and stored teeth

From the Institute for Forensic Medicine at the University of Würzburg
Board of Directors: Prof. Dr. med. Christoph Meissner


Inaugural - Dissertation to obtain a doctorate from
Medical Faculty of the Julius Maximilians University of Würzburg
presented by
Sebastian Gebhard
from Schnaittach
Würzburg, July 2009
Speaker: Prof. Dr. med. Dieter Patzelt
Co-referee: Prof. Dr. med. dent. Dipl.-Ing. Ernst-Jürgen Richter
Dean: Prof. Dr. med. Matthias Frosch
Oral exam day: March 11, 2010
The doctoral candidate is a dentist.

 


Table of Contents
1 Introduction
1.1 Forensic Dentistry
1.2 Molecular biological principles
1.2.1 The genetic fingerprint
1.2.2 Identification options by means of DNA analysis
1.2.2.1 Variable number of tandem repeats (VNTR)
1.2.2.2 Restriction Fragment Length Polymorphisms (RFLP)
1.2.2.3 Short tandem repeats (STR)
1.2.2.4 Mitochondrial DNA (mtDNA)
1.2.2.5 Sex differentiation using amelogenin
1.3 General dental principles
1.4 Task
2 materials and methods
2.1 Instruments and reagents
2.1.1 General consumables and devices
2.1.2 Tooth cleaning and sampling
2.1.3 DNA extraction
2.1.4 PCR
2.1.5 Polyacrylamide Gel Electrophoresis (PAGE)
2.1.6 High resolution capillary electrophoresis:
2.2 Tooth samples and storage
2.2.1 Teeth from the dental practice of Dr. Werner Gebhard and Dr. Lothar Gebhard
2.2.2 Teeth from the maxillofacial surgery practice of Dr. Dr. Hemmerich
2.2.3 Teeth of the Natural History Society Nuremberg
2.2.3.1 Teeth from an excavation in Katzwang
2.2.3.2 Teeth from the Dietersberg cave
2.2.4 Teeth from dissected material
2.3 Experimental set-ups
2.3.1 Freshly extracted teeth
2.3.2 Teeth buried in the ground
2.3.3 Teeth stored in water
2.3.4 Teeth exposed to sunlight
2.3.5 Teeth made from dissected material
2.3.6 Teeth exposed to intense heat
2.3.7 Teeth extracted 28 and 29 years ago
2.3.8 Teeth from the excavation in Katzwang
2.3.9 Teeth from the Dietersberg cave
2.4 Methods
2.4.1 Avoiding contamination
2.4.2 Sampling
2.4.3 DNA extraction
2.4.4 Polymerase Chain Reaction (PCR)
2.4.4.1 STR SE33
2.4.4.2 AmpFlSTR® SGM Plus Kit
2.4.4.3 STR TPOX-vs
2.4.4.4 Mentype® NonaplexQS PCR Amplification Kit
2.4.5 Gel electrophoresis and silver staining
2.4.6 High resolution capillary electrophoresis
2.4.7 Quantifiler® Human DNA Quantification Kit
3 results
3.1 Freshly extracted teeth
3.2 Teeth buried in the ground
3.3 Teeth stored in water
3.4 Teeth exposed to sunlight
3.5 teeth made of dissected material
3.6 Teeth exposed to intense heat
3.7 Teeth extracted 28 and 29 years ago
3.9 Teeth from the Dietersberg cave
4 discussion
4.1 Methods
4.1.1 Sampling
4.1.2 DNA extraction
4.1.3 PCR
4.1.4 Real-Time-Quantitative-PCR (RTQ-PCR)
4.2 Complications and PCR Artifacts
4.2.1 "allelic dropout"
4.2.2 Incorrect gender statement
4.2.3 Stuttering gangs
4.3 Results
4.3.1 Freshly extracted teeth
4.3.2 Teeth buried in the ground
4.3.3 Teeth stored in water
4.3.4 Teeth exposed to sunlight
4.3.5 Teeth made from dissected material
4.3.6 Teeth exposed to intense heat
4.3.7 Teeth extracted 28 and 29 years ago
4.3.8 Teeth from the excavation in Katzwang
4.3.9 Teeth from the Dietersberg cave
4.4 DNA analysis of archaeological skeletal finds
5 Summary
6 Bibliography


List of figures
Fig. 1: Lengths of the analysis products in relation to the fragment length of the
degraded DNA (from Hummel 2003, p. 28)
Fig. 2: Apicales delta and medullary canal (Safe et al. 1928, p. 128)
Fig. 3: The entrance to the Dietersberg cave
Fig. 4: Location of the teeth buried in the ground
Fig. 5: Location of the teeth stored in water
Fig. 6: Location of the teeth exposed to solar radiation
Fig. 7: Storage vessel for teeth that are almost 30 years old
Fig. 8: Skull with sample tooth No. 40 with trephination drilling
Fig. 9: The three teeth from the Dietersberg cave (samples 48-50)
Fig. 10: Tooth with a circular groove in front of the pulp opening
Fig. 11: Sampling location
Fig. 12: Trial tooth after breaking open
Fig. 13: Polyacrylamide gel on the cooling plate
Fig. 14: Stained pre-gel of samples 51 and 52; left: the SE33, right: SGMMultiplex;
both with ladder, neutral sample, positive and negative control
Fig. 15: Upper graphic: The reference ladder of this system is used for
Comparison with the sample results. Middle graphic: One not
clearly detectable result. The two peaks of sample 26
are in the range of alleles 16 and 17, but only occur a lot
weakly stand out from the background noise and are therefore associated with a
"?" Marked. Lower graphic: Sample 47 is a clear one
Allele assignment possible.
Fig. 16: Deeply destroyed tooth of sample no.9
Fig. 17: Example of an electropherogram of the Mentype® NonaplexQS analyzed
on the ABI PRISM® 310 analyzer with red marked DNA size standard
550 (ROX) (Biotype AG, Mentype® product information
NonaplexQS)
Fig. 18: Stutter bands in di- and tetranucleotide STR amplification products
(from Hummel 2003, p. 36)


List of tables
Tab. 1: Overview of the raw material used by Dr. Werner
Gebhard and Dr. Lothar Gebhard
Tab. 2: Overview of the starting material of the maxillofacial surgery practice Dr. Dr.
Hemmerich
Tab. 3: Overview of the original material from the excavation near Katzwang
Tab. 4: Overview of the starting material from the Dietersberg cave
Tab. 5: Overview of the starting material from the section material of the
Institute for Forensic Medicine Würzburg
Tab. 6: PCR approach for SE33
Tab. 7: PCR conditions for SE33
Tab. 8: Systems of the AmpFlSTR® SGM Plus® (according to instructions
AmpFlSTR® SGM Plus®, Applied Biosystems)
Tab. 9: PCR approach for AmpFlSTR® SGM Plus® (according to instructions
AmpFlSTR® SGM Plus®, Applied Biosystems)
Tab. 10: PCR conditions for AmpFlSTR® SGM Plus® (according to instructions
AmpFlSTR® SGM Plus®, Applied Biosystems)
Tab. 11: PCR approach for TPOX-vs (Hellmann et al. 2001)
Tab. 12: PCR conditions for TPOX-vs (Hellmann et al. 2001)
Tab. 13: Systems of the Mentype® NonaplexQS (according to the instructions for Mentype®
NonaplexQS, Biotype® and Homepage Biotype AG)
Tab. 14: PCR approach for Mentype® NonaplexQS (according to instructions for
Mentype® NonaplexQS, Biotype®)
Tab. 15: PCR conditions for Mentype® NonaplexQS (according to instructions for
Mentype® NonaplexQS, Biotype®)
Tab. 16: Approach for capillary electrophoresis
Tab. 17: Preparation for Quantifiler® Human DNA Quantification Kit (according to instructions
Quantifiler® Human DNA Quantification Kit, Applied Biosystems)
Table 18 (a): Quantification, SE33 and SGM of the freshly extracted teeth
Table 18 (b): TPOX-vs and Nonaplex of the freshly extracted teeth
Tab. 19 (a): Quantification, SE33 and SGM of the buried teeth
Table 19 (b): TPOX-vs and Nonaplex of the buried teeth
Tab. 20 (a): Quantification, SE33 and SGM of the teeth stored in water
Tab. 20 (b): TPOX-vs and Nonaplex of the teeth stored in water
Tab. 21 (a): Quantification, SE33 and SGM exposed to solar radiation
teeth
Tab. 21 (b): TPOX-vs and Nonaplex teeth exposed to sunlight
Tab. 22 (a): Quantification, SE33 and SGM of the teeth from the section material
Tab. 22 (b): TPOX-vs and Nonaplex of the teeth from the section material
Tab. 23 (a): Quantification, SE33 and SGM of teeth exposed to strong heat
Tab. 23 (b): TPOX-vs and Nonaplex of teeth exposed to intense heat
Tab. 24: Quantification, SE33 and SGM of those extracted 28 and 29 years ago, respectively
teeth
Tab. 25 (a): Quantification, SE33 and SGM of the teeth of the excavation in
Katzwang
Tab. 25 (b): TPOX-vs and Nonaplex of the teeth from the excavation in Katzwang
Tab. 26: Quantification, TPOX-vs and Nonaplex of the teeth from the Dietersberg cave


List of abbreviations
6-FAM: 6-carboxy fluoresein
AKFOS: Forensic Odonto-Stomatology Working Group
bp: base pair
DNA: deoxyribonucleic acid
BSA: Bovine serum albumin
dNTP: deoxynucleoside triphosphate
HEX: 4,7,2 ', 4', 5 ', 7' -hexachloro-6-carboxy-fluorescein
NaOCl: sodium hypochlorite
NHG: Natural History Society Nuremberg
OK: upper jaw
PAGE: polyacrylamide gel electrophoresis
PCR: polymerase chain reaction
ROX: 6-carboxy-X-rodamine
RTQ-PCR: Real-Time-Quantitative-PCR
STR: short tandem repeats
Lower jaw: lower jaw
VNTR: variable number of tandem repeats
vs-STR: very short STR
WF: root canal filling

 



1 Introduction
1.1 Forensic Dentistry
The assistance of dentists in examining teeth for identification is essential
not uncommon in forensic medicine. In 1976, during the 102nd
Annual meeting of the German Society for Dentistry, Oral and Maxillofacial Medicine of
Working group Forensic Odonto-Stomatology (AKFOS) founded, which is responsible for
Task has made “the forensic aspects in dentistry, oral and maxillofacial medicine
to sift through and to the scientific interests of this area [...]
promote ”(§3 of the statutes of the AKFOS).
A forensic dentist will always be consulted when using other methods
to identify an unknown corpse can no longer be realized
or were unsuccessful. Such identification is usually through a
Survey of the dental status of a dead person or by means of an X-ray film and then
Comparison with the dental documents of the person in question
Person possible. Not just the number of missing teeth, placed implants and restoration
with root canal treatments and fillings, but also their position
in the jaw, its extent and the materials used can
human teeth as good as unmistakable. The supply of fixed
or removable, prosthetic or orthodontic works (crowns,
Bridges, telescopic, model cast, clasp, cover denture prostheses, total prostheses,
Multiband appliances, braces, ...), surgical interventions (root tip resections,
Bone augmentations, sinus lifts, ...), the presence of excess
or dislocated teeth and changes due to age or pathological
Processes (tooth tilting, atrophy of the alveolar ridges, cysts, ...) expand
also the inter-individual variability (Wittacker et al. 1993, p. 9ff; Rötzscher 2003,
P. 1590ff).
It becomes more difficult when there are no remaining teeth. In some
It is therefore common in countries for prostheses to be marked with the
provide an identification aid (Rötzscher 2000, p. 157f).
But the forensic dentist is not only in for the identification of people
the situation. Age estimation is also possible with teeth. Based on
Chronology of tooth development and growth can take place in children
14 years, the age can be determined with a reliability of +/- 2 years.
Resorption of the milk tooth roots, as well as formation and calcification are im
X-ray features that are easy to display.
The degree of racemization of aspartic acid is suitable for any age as one
precise possibility of age estimation. Because of its extremely bradytrophic metabolism
Dentine is an ideal tissue for biochemical age determination.
Aspartic acid occurs in two forms: in an L and in one
D shape. In the biosynthesis of human proteins, only L-amino acids are used
educated. From the moment of synthesis, there are spontaneous, not
enzymatic conversions to the D-form. This in vivo racemization leaves
prove themselves in the dentist after appropriate preparation and analysis. since
In 1995 this method was also used in Germany for living people
(Ritz-Timme et al. 2003).
Extracted teeth allow the age to be estimated by determining the translucency
of the root dentine (Bang et al. 1970).
One of the greatest challenges facing forensic dentists so far has certainly been
the tsunami disaster in the Indian Ocean in December 2004. Like Petju,
Schuller-Gotzburg or Tan explain in their studies, all were German victims
identified in Thailand by dental records. Problems due to lack of
However, documents were available for the identification of local victims (Petju et al.
2007; Schuller-Gotzburg et al. 2006; Tan 2005).
But not only natural disasters such as earthquakes, floods or volcanic eruptions
repeatedly demand the use of forensic dentists. Also
Disasters such as the train accident in Eschede, ship accidents or plane crashes
make their use just as necessary. Terrorist attacks like in September 2001
in the USA, are also expanding the field of application of forensic dentistry
(Glass 2005).
Developed by the Victorian Institute of Forensic Medicine in Melbourne, Australia
since 1997 a computer-aided software that automatically compares the tooth status
and can examine for matches. This “Disaster And Victim
IDentification “program (DAVID) has been a web-based program since 2005
reprogrammed, which will then be quickly usable worldwide (Clement et al.
2006).
Teeth in particular are often very suitable for creating what is known as genetic
Fingerprints, as they are very resistant to external influences.
“They are with high-grade tissue destruction such as water storage, putrefaction or
Corpse burn is a relatively reliable source of sufficiently high molecular weight DNA.
Due to its localization, the tooth pulp is well protected from degradation and contamination
protected ”(Patzelt et al. 2003, p. 1045).
1.2 Molecular biological principles
1.2.1 The genetic fingerprint
In Europe, the has been used to identify people since the beginning of the 20th century
“Normal” fingerprints, the dactylogram, have become indispensable. With this one
Procedure you make the individual skin ridge pattern of the fingertip
Use (Siegmund-Schultze 2003).
In 1985 Jeffreys recognized the potential of individual-specific repetitive DNA as
Basis for identification. By analyzing these non-coding sections of DNA
it is possible for each individual with a unique, barcode-like one
Associating patterns. This procedure is called "genetic
Fingerprint ”because, similar to the dactylogram, it is a unique one
Individual assignment is possible. The only exception to this are identical twins.
With the techniques of that time, however, was a large amount of high molecular weight
DNA required (Jeffreys et al. 1985).
1.2.2 Identification options by means of DNA analysis
The "Human Genome Project" found that only about 5% of the
human genome are responsible for coding the proteins. They don't
coding sections are interesting for identification because they contain a
show interindividual variability. Different types of polymorphic DNA
Structures are now known which are used in the search for clues
(Hummel 2003, p. 26f).
1.2.2.1 Variable number of tandem repeats (VNTR)
Initially, longer, so-called VNTRs (variable number of tandem repeats)
or mini satellites are used for forensic investigations. This repetitive
DNA sequences consist of 5-50 repeats of 20-70 bp in length
Segments. As a result, you need high molecular weight DNA for this. Special oligonucleotides,
so-called primers, set the starting point of the amplification and
thus define which locus on the DNA is amplified in the PCR (Horn et
al. 1989).
1.2.2.2 Restriction Fragment Length Polymorphisms (RFLP)
In the RFLP analysis (restriction fragment length polymorphisms) of PCR products
double-stranded DNA is produced by restriction enzymes, so-called endonucleases,
digested and then separated by electrophoresis. With
Special probes can now mark the resulting bands and make them visible
be made. This banding pattern is different for each individual and
can also be used for identification. The disadvantage of this method
is that there must be a large amount of high molecular weight DNA,
which limits its use in forensic medicine (Hummel 2003, p. 126f).
1.2.2.3 Short tandem repeats (STR)
From the beginning of the 1990s, shorter, polymorphic structures replaced the VNTRs.
For the analysis of these microsatellites or STRs (Short Tandem Repeats) it was sufficient
now shorter DNA fragments and a more degraded DNA could be used
Individual assignment can be used. The repeating core sequence is
only 2-6 bp in size, so that the gene locations to be analyzed are only 100-300 bp long
(Hammond et al. 1994; Urquhart et al. 1995).
With the development of new primers, even smaller target fragments could be captured
and thus even more degraded DNA can be used. This was possible by
the annealing position of the primers can be brought closer to the repetitive sequence
could. In the vs-STRs (very short STRs) it was possible to reduce the allele sizes to below
87 bp for the TPOX system, below 86 bp for the TH01 system and below 106 bp for the
Lower system FES. When comparing the classic system with the vs-
System of the same individual is a match of allele numbers too
expect (Hellmann et al. 2001).
The following scheme relates the length of the analysis products to the
Fragment length of the degraded DNA:
Fig. 1: Lengths of the analysis products in relation
the fragment length of the degraded DNA (from
Hummel 2003, p. 28)
1.2.2.4 Mitochondrial DNA (mtDNA)
In contrast to chromosomal DNA, which is half from the mother and half from the
the other half is inherited from the father, the mitochondrial DNA is exclusively in
passed down the maternal line. This is because the oocyte has the
complete set of mitochondria already included and from the spermatozoa
Fertilization usually does not add any more. All family members of the
The maternal line consequently have the same mtDNA sequence (Hühne et al. 1999).
Within the circular mtDNA, most of the sequence polymorphisms come into
the "d-loop control region", which is mainly made up of regions I and II
(hypervariable region I and II). Using sequence analysis of DNA
these regions of an unknown person and comparison with DNA from in question
Persons coming from or from their maternal relatives can be identified
carried out (Bär et al. 2000).
In addition to the high variability, the mtDNA offers advantages such as the analysis of family relationships
the maternal line and the occurrence of a high one
Number of copies of 103-104 per cell (Geserick et al. 1998).
1.2.2.5 Sex differentiation using amelogenin
Amelogenin is a protein that plays a role in the enamel formation of the teeth
plays. It is located on the two gonosomal chromosomes X and Y in places
Coded Xp22.31-p22.1 and Yp11.2. It was discovered in the early 1990s that the
The first intron of this gene on the X chromosome is 6 bp shorter and instead of 112 bp only
106 bp. Thus, a band (106 bp) is indicative of the analysis
female, two bands (106 bp and 112 bp) on one male
Gender (Mannucci et al. 1994).
1.3 General dental principles
A person with wisdom teeth has a total of 32 permanent teeth, of which per
Jaw quadrant two incisors, one canine, two small ones
Back teeth (premolars) and three large molars (molars). The basic one
The anatomical structure is the same for all teeth (Fischer 1935, p. 2).
The numbering of the teeth in this work was based on that of the Federation
Dentaire Internationale introduced the so-called FDI scheme in 1970.
This two-digit tooth scheme consists of the number of the quadrant and
the tooth numbered consecutively from the center (Lehmann et al.
2002, p. 9).
A tooth consists of three hard tissues, the dentin, the tooth enamel
(Enamelum) and the dental cement (Cementum).
The dentin consists of an organic, collagen-rich basic substance (e.g.
70%) and for hardening from inorganic hydroxylapatite crystals Ca5 (PO4) 3 (OH)
(about 30%). In contrast to bone formation, in which the osteoblasts are in
wall in the bone, the dentin-forming cells, the odontoblasts, remain on
The edge of the dentin lie and only send out appendages 1-3 μm thick
("Tomes fibers") into the dentin tubes (tubuli dentales).
To protect against wear and tear, the dentin is made of the hardest human tissue,
the enamel, coated. Similar to bone or dentine, this consists of
made of hydroxyapatite crystals that are embedded in an organic base substance.
The organic share is less than 1%. The enamel-forming cells that
Ameloblasts, lie on the outside of the tooth enamel and become after the tooth eruption
quickly chewed. Tooth enamel defects are therefore not capable of regeneration.
The part embedded in the bone is covered with a thin coating of bone-like material
Surrounding dental cement. Strong belt pulls pull from him into the
Jawbone and thus anchor the tooth in the tooth cavity, the alveolus (Lippert
2003, p. 601).
Inside the tooth, the pulp cavity, there is a nerve and vascular structure
Tissue, which, among other things, consists of undifferentiated cells, fibroblasts and
Odontoblast exists (Hellwig et al. 2003, p. 248).
As early as 1928 it was known that the root canal was not only
tapered to finally in the root tip in the foramen
apicale, but usually just before the
The exit is divided into several branches and thus an apical delta
forms (see Fig. 2). Make another form of secondary branches
Medullary canals that perforate a side wall of the root
can and open on the outside (Safe et al. 1928,
P. 128f).
Each tooth in the dentition is characterized by its root feature, mass feature, angle feature
and other dental features clearly identify its position in the dental arch
assignable. The root feature is defined as the distal deviation of the root
in relation to the tooth axis. The mass feature means that the teeth are mesial
are bulkier than distal. The angle feature says that seen from the vestibular
the angle of the incisal edge with the approximal surface is more acute mesially than distal
(Lehmann et al. 2002, p. 13).
Fig. 2: Apicales delta
and medullary canal (Sure
et al. 1928, p. 128)
In this work tissue of the tooth pulp was examined. The pulp tissue is
but not always healthy. As a result of tooth decay, one often develops
Pulpitis. This pulp tissue modified in this way is subject to inflammation
usual cardinal symptoms rubor, tumor, calor, dolor and functio laesa. Indeed
the particular topography causes characteristic lice forms. By
Stimulus-induced hyperemia leads to an increase in tissue pressure.
As a result, leukocytes migrate through chemokinetic and chemotactic factors
from the vessels to the reaction site; reversible pulpitis develops. Can he
Stimulus are removed, healing occurs. If the stimulus persists
short-lived neutrophilic granulocytes migrate, which die when they die
release cell-toxic components and proteolytic enzymes, which when appropriate
Concentration inevitably leads to pulp necrosis.
In addition to aseptic, inflammation-related necrosis, a
Colliquational necrosis is caused by bacteria invading the pulp. There
the bacterial infestation can either be caused by a carious lesion of the hard tissue of the tooth
take place or the bacteria arrive retrograde through an inflammation of the tooth supporting apparatus
(Periodontitis) via the apical foramen into the endodontium (paro-
Endo lesion) (Hellwig et al. 2003, p. 254ff).
The treatment of pulpitis consists in a root canal treatment. Here the
pulpic or gangrenous crown and root pulp removed, along with the canal lumen
endodontic instruments and then the root canal with
Disinfects irrigation solutions and medical insoles. When the channels (sometimes only
after several sessions) are symptom-free and can be dried out,
the root canals are closed with gutta-percha points and a root canal filling paste
(Weber 2003, p. 298ff).
Because of the commonly used disinfecting root canal inserts, it is questionable whether
DNA that can be used for analysis can be obtained from root-filled teeth
can. Specially performed rinses with sodium hypochlorite to clean the
Root canals destroy existing DNA.
1.4 Task
The aim of this doctoral thesis was to check to what extent the further development
molecular detection methods in recent years have opened up new possibilities for
Typing of STR systems on teeth. In particular, the
Investigated how different storage conditions affect the
Impact degradation of pulpal DNA. For this some were at the time of
Tooth extraction vital teeth buried in the ground, stored in water, exposed to sunlight
exposed and heated in the heating cabinet. Two teeth from the
Sections of the Institute for Forensic Medicine of the University of Würzburg were without
special storage used in this work. Teeth with existing root canal fillings
were analyzed for usable results. Additionally stood in front
Teeth extracted 28 and 29 years ago, as well as 380-550 years and 2,300-2,800 years
old teeth available for examination.


2 materials and methods
2.1 Instruments and reagents
2.1.1 General consumables and devices
Premium freezer Liebherr, Biberach
Bosch refrigerator, Stuttgart
Pipette tips Eppendorf, Hamburg
Safeguard filter tips 10, 30, 100, 200 and 1000 μl PeqLab, Erlangen
Vortex device Genie 2 Bender & Hobein, Zurich
Warming cabinet model 600 Memmert, Schwabach
2.1.2 Tooth cleaning and sampling
K-File Colorinox®, sizes 20 - 55 Dentsply, Constance
Petri dish, sterile Hartenstein, Würzburg
NaOCl Roth, Karlsruhe
Lever to Bein Aesculap, Tuttlingen
Premolar forceps Aesculap, Tuttlingen
Diamond drill Komet, Lemgo
Turbine Kavo, Biberach
Weld-in foils Hawo, Obringheim
Tweezers Carl Martin, Solingen
2.1.3 DNA extraction
E.Z.N.A.® Tissue DNA Mini Kit PeqLab, Erlangen
Ethanol, absolute Roth, Karlsruhe
Water bath model B Lauda, ​​Königshofen
Centrifuge Microliter Hettich, Tuttlingen
Incubator B 30 Memmert, Schwabach
2.1.4 PCR
Microcon YM-30 Millipore, Eschborn
PCR water LiChrosolv® Merck, Darmstadt
Mineral oil Sigma, Steinheim
Thermocycler ABI Gene Amp PCR System 2400 Applied Biosystems, Weiterstadt
Thermal cycler “Personal Cycler” Biometra, Göttingen
Amplification with the "AmpFl STR ® SGM Plus" kit:
AmpliTaq Gold® (5 U / μl) Applied Biosystems, Weiterstadt
AmpF1STR® PCR Reaction Mix Applied Biosystems, Weiterstadt
AmpF1STR® Profiler Plus Primer Set Applied Biosystems, Weiterstadt
Amplification of the SE33 system:
10x Taq Buffer Eppendorf, Hamburg
Nucleotide triphosphate (5 mM) Pharmacia, Freiburg
MgCl solution (2 mM) Applied Biosystems, Weiterstadt
Taq DNA polymerase (5 U / μl) Eppendorf, Hamburg
Primer 1 and 2 (SE33) Applied Biosystems, Weiterstadt
Amplification with the Mentype ® Nonaplex QS PCR Amplification Kit:
JumpStartTM Taq DNA Polymerase Sigma-Aldrich, Steinheim
Reaction Mix A Biotype, Dresden
Primer Mix Biotype, Dresden
Amplification of the TPOX-vs-System:
like SE33 system
Primer 1 and 2 MWG-Biotech, Ebersdorf
Quantifiler ® Human DNA Quantification Kit
Quantifiler® PCR Reaction Mix Applied Biosystems, Weiterstadt
Quantifiler® Human Primer Mix Applied Biosystems, Weiterstadt
T10E0.1 (10 mM Tris-HCl [pH 8.0], 0.1 mM Na2EDTA) Applied Biosystems, Weiterstadt
PRISM® 7000 Sequence Detection System Applied Biosystems, Weiterstadt
2.1.5 Polyacrylamide Gel Electrophoresis (PAGE)
Low resolution horizontal polyacrylamide gel electrophoresis (PAGE):
Approach for a 5% polyacrylamide gel:
16 ml H2O
2.1 ml 10 x TBE buffer
2.7 ml acrylamide / bisacrylamide solution
(Acrylamide-Bis 19: 1) Serva, Heidelberg
200 μl APS Serva, Heidelberg
1.5 μl TEMED Serva, Heidelberg
Glass plates 124 x 258 mm glass wedge, Würzburg
Sample buffer:
0.25% bromophenol Blau Roth, Karlsruhe
0.25% xylene cyanol Roth, Karlsruhe
25% Ficoll 400 Roth, Karlsruhe
10 x TBE buffer:
890 mM Tris-Borate Roth, Karlsruhe
25 mM EDTA (pH 8.0) Roth, Karlsruhe
Sample application plate Serva, Heidelberg
Gene Ruler 100 bp Ladder Plus MBI Fermentas, Lithuania
Nitric acid 1% Roth, Karlsruhe
Silver nitrate solution:
0.4 g AgNO3 Roth, Karlsruhe
200 ml distilled water.
Developer solution:
0.28 M NaH2CO3 Roth, Karlsruhe
0.019% formalin Roth, Karlsruhe
Acetic acid 10% Roth, Karlsruhe
Glycerol Roth, Karlruhe
Electrode strips Serva, Heidelberg
Electrophoresis chamber LKB 2117 Multiphor II LKB / Pharmacia, Freiburg
Electrophoresis cooler LKB 2219 Multitemp II LKB / Pharmacia, Freiburg
GelBond PAG-Film Pharmacia, Freiburg
Voltage device LKB 2297 Macrodrive 5 LKB / Pharmacia, Freiburg
Orbital shaker Polymax 2040 Heidolph, Schwabach
Foil for Gel Pharmacia, Freiburg
2.1.6 High resolution capillary electrophoresis:
GeneScan 500 ROX Size Standard Applied Biosystems, Weiterstadt
Formamid Sigma, Steinheim
Genetic Analyzer ABI 310 Applied Biosystems, Weiterstadt
2.2 Tooth samples and storage
The test material for this work comprises a total of 50 teeth and two
Gauze swabs used to stop bleeding after tooth extraction (No. 46
and 47). For the most part (39 teeth) these teeth come from dental practices
by Dr. Werner Gebhard, Schnaittach, and Dr. Lothar Gebhard, Nuremberg.
Teeth nos. 21 to 23 come from the maxillofacial surgery practice of Dr. Dr. Hemmerich,
Munich. Teeth No. 38 to 40 were from the Natural History Society
Nuremberg (NHG) from the Department of Prehistory of Dr. Mühldorfer from one
Excavation in Katzwang near Nuremberg left for investigations. At a
The teeth of specimens 48, 49 and 50 were excavated in the Dietersberg cave
found and also provided by the NHG Nuremberg for this work. Out
The teeth of the section of the Institute for Forensic Medicine in Würzburg were lost
Nos. 33 and 34 taken.
After completion of the respective experiment, the teeth were kept in the institute at -20 ° C
Frozen until further use. The DNA degradation that takes place
was classified as negligible.
2.2.1 Teeth from the dental practice of Dr. Werner Gebhard and Dr. Lothar Gebhard
Granulation and periodontal tissue were removed from extracted teeth. To
Drying, these were sealed in shrink-wrap and given to the author.
Before being used, they became dark, dry and cool for a short time
stored.
The patients were informed about the planned work and gave their consent.
Time of extraction, age and gender were recorded anonymously and
used for work.
The following table gives an overview of the starting material of these
both practices again:
Sample no.
Age of
Patient Genus Tooth Extraction Day Comment
1 19 m 18 May 20, 2005
2 84 m 42 05/30/2005 Tooth vital
3 54 m 12 June 3, 2005 Tooth vital
4 69 m 31 06/02/2005 Tooth vital
5 69 m 42 06/02/2005 Tooth vital
6 80 w 23 06.06.2005 Zahn avital
7 74 w 26 07/05/2005 deeply destroyed
8 51 m 23 May 17, 2005 deeply destroyed
9 51 m 25 May 17, 2005 deeply destroyed
10 64 w 36 06/15/2005
11 51 m 24 May 17, 2005 deeply destroyed
12 21 m 28 June 8, 2005
13 21 m 38 06/08/2005
14 87 w 24 08/19/2005 severely sclerosed
15 18 w 28 10/28/2003 wide open apical foramn
16 48 m 23 08/04/2005 Swab No. 47
17 79 w 42 06/06/2005
18 18 m 28 June 2, 2005
19 83 w 13 June 15, 2005
20 69 m 46 07/04/2005 Tooth vital
24 35 m 26 08/18/2005 WF on 08/02/04
25 57 m 26 May 19, 2005 WF since ~ 6 a
26 58 w 37 07/07/2005 Swab No. 46
27 51 m 18 July 30, 2005
28 59 m 12 August 2, 2005
29 57 w 38 07/22/2005 Tooth vital
30 55 m 34 08/02/2005
31 69 m 38 07/07/2005
32 49 m 38 07/08/2005 Tooth vital
35 38 m 28 August 25, 2005
36 19 m 38 06/16/2005 Tooth vital
37 59 m 11 13.10.2005
41 53 w 41 09/22/2005 Tooth vital
42 44 w 17 04.10.2005 Tooth vital
43 44 w 48 06/17/2005 Tooth vital
44 19 m 28 06/16/2005 Tooth vital
45 69 m 36 07/07/2005 Tooth vital
51 23 w 38 11/06/2006 Tooth vital
52 45 w 48 12/27/2006
Tab. 1: Overview of the raw material used by Dr. Werner Gebhard and Dr. Lothar
Gebhard
2.2.2 Teeth from the maxillofacial surgery practice of Dr. Dr. Hemmerich
The teeth from the maxillofacial surgery practice Dr. Dr. Hemmerich, Munich, come from
a collection of teeth with abnormal roots that Dr. Werner
Gebhard removed it during his assistantship in 1977/78. you are
consequently it was drawn 28 and 29 years ago. After tooth extraction, they were
cleaned and bleached with H2O2. They were then stored at room temperature
dry in a glass container (see Fig. 7, p. 22). The age and gender
the patient could no longer determine after such a long period of time
become. The position in the dental arch was determined on the basis of the crown shape, crown alignment,
Curvature feature and angle feature (see Chapter 1.3, p. 7) assigned. The
Teeth were completely preserved and without carious lesions (see Table 2).
Sample no. Age of the patient Genus Tooth Extraction year Remark
21 ? ? 23 1976/77
22 ? ? 48 1976/77
23 ? ? 23 1976/77
Tab. 2: Overview of the starting material of the maxillofacial surgery practice Dr. Dr. Hemmerich
2.2.3 Teeth of the Natural History Society Nuremberg
A total of six teeth from two epochs were taken from the natural history
Society Nuremberg (NHG) made available for investigation purposes.
These were recovered during two archaeological excavations and from
the NHG has already been scientifically processed.
Two teeth were still anchored in the jawbone and their position in the dental arch
could thus be clearly identified. The other four teeth lay as single teeth
and were able to use typical tooth features, as already in Chapter
1.3, p. 7, assigned to the position in the dental arch.
2.2.3.1 Teeth from an excavation in Katzwang
Three teeth come from an excavation in 2004 at the cemetery and
Defensive wall of the church "Our Lady" in Katzwang in the south of Nuremberg.
The associated skeletons were about 70 cm outside the cemetery wall
discovered below the current grass surface. In total, skeletal parts of
13 individuals found, whereby it can be assumed that this is a
must act larger burial place. The human corpses were after
Christian custom in east-west direction and buried with crossed arms. At
Various bones have been found to have injuries from cutting and stabbing weapons
become. It is currently believed that the bones consist of one of the following three
Wars should come from:
- 1st Margrave War (1449/50)
- 2nd Margrave War (1552-54)
- Thirty Years War (especially 1631/32).
Consequently, an age of the bone finds of around 380-550 years comes into consideration.
A radiocarbon analysis for more precise dating is still pending, but is
planned after a planned anthropological study (Zeitler 2005).
The following table provides an overview of this starting material:
Sample no.
Age of
Corpse Genus Tooth Year of excavation Comment
38? ? 18 2004 single tooth
39? ? 17 or 18 2004 single tooth
40? ? 16 2004 tooth in the jaw
Tab. 3: Overview of the original material from the excavation near Katzwang
2.2.3.2 Teeth from the Dietersberg cave
Three other individual teeth examined, also
from the inventory of the NHG Nürnberg
from an excavation of the archaeologist J. R. Erl in the
Dietersberg cave near Egloffstein. In addition to numerous
Skull and skeletal fragments of
adults, young children and fetuses
there were also 44 loose teeth (Erl 1953,
P. 238).
The cave is an 11 m deep shaft crevice with
an approximately 1.5 m x 1.0 m entry hole
(see Fig. 3). The cone of rubble at the bottom of the
Cave consisted of humus sediment, which
got soaked in rain.
This cave probably served as a burial place for a family association
(epigenetic features indicate family relationships) of forest and
Fig. 3: The entrance to the Dietersberg cave
Wild beekeeping from the late Hallstatt period (HaC and HaD) and the early La Tène period,
both epochs of the Iron Age. This could be from grave goods and jewelry
getting closed. The age of the teeth is therefore 2,300-2,800 years.
Forensic-anthropological studies have shown that there are remains of
have found at least 46 individuals in the cave. Under the main inventory number
8289 they are kept in the collection of the NHG (Baum 1999).
The exact storage conditions of the excavation remains can no longer be determined
because during the war years the collection items were used to protect against
Looting has been outsourced several times. Until the NHG moved in
In 2000 the preparations were finally stored in the attic and were there all the time
exposed to changing temperature and humidity. Only then could they
Basement rooms of the Norishalle can be used for storage (Mühldorfer).
Since very many of the excavated teeth had deep carious lesions (the
Caries incidence was 33.5%), three externally intact teeth were used for this
Investigation used. Upon successful creation of a genetic fingerprint
would be family relationships and the gender of the
Individuals of great archaeological importance. The NHG would then be on more
Investigations have been interested.
The following table provides information about the teeth from the Dietersberg cave
summarized:
Sample no. Age of
Corpse Genus Tooth Year of excavation Comment
48? ? 11 1928 single tooth
49? ? 23 1928 single tooth
50? ? 18 1928 single tooth
Tab. 4: Overview of the starting material from the Dietersberg cave
2.2.4 Teeth from dissected material
In two cases, teeth could be seen from cadavers with lay times of 14 to 17 days
the section material of the Institute for Forensic Medicine Würzburg used for the investigation
become.
Sample tooth no.33 became on 08/30/05, sample tooth no.34 on 08/31/05 from the
Corpses extracted. The teeth were kept in sterile condition until further processing
The tubes are stored at -20 ° C in the institute's own freezer.
Table 5 provides information about this starting material:
Sample no. Age of deceased Genus Tooth Lying time Post-mortem day Comment
33 36 m 33 ~ 17d 08/25/2005
34 59 m 33 ~ 14d 08/31/2005
Tab. 5: Overview of the starting material from the section material of the Institute for Forensic Medicine
Wurzburg
2.3 Experimental set-ups
2.3.1 Freshly extracted teeth
To become familiar with the equipment and reagents and to check
whether the E.Z.N.A.® Tissue DNA Mini Kit is suitable for this analysis task,
some teeth were examined by DNA analysis without storage. Furthermore were
asensible teeth, teeth with obliterated pulp cavity, wisdom teeth with wide
open apical foramen and teeth with large carious lesions or root canal treatments
analyzed.
These teeth were not stored separately, but were evaluated directly.
Like all other teeth, they were kept at -20 ° C until the evaluation
stored in the freezer.
2.3.2 Teeth buried in the ground
In this experimental set-up, single-use plastic cups were used
provided with holes on the bottom and with
Labeled with foil pen. These cups were used
to find and identify the teeth again
can. The holes were used to ensure that after
Do not dump water in the cup when it rains
could. Each cup became one-third with soil
filled, then a tooth is placed and earth is again placed on it up to the edge of the cup.
Fig. 4: Location of the in the ground
buried teeth
pours. The cups were then placed in a relatively damp and shady area
Bury it under bushes to the top. The teeth were thus about 4 cm
underground and were exposed to the weather conditions (see Fig. 4,
P. 19).
For the examinations, teeth were after about four weeks (tooth 10), about four
Months (tooth 26), about half a year (teeth 31 and 32) and about a year
(Teeth 43, 44 and 45) lying time taken.
2.3.3 Teeth stored in water
The five teeth stored in water (No. 35, 41, 42,
36 and 37) were in small, with tap water
given filled glass vessels. The lids were
not screwed, but just laid on to one
To enable gas exchange (see Fig. 5). Regularly
the water level was checked and at
Refilled with water as required. This series of experiments
stored in the boiler room at temperatures around 20 ° C.
Teeth were removed after about three months (tooth 35), half a year
Year (teeth 41 and 42) and one year (teeth 36 and 37).
2.3.4 Teeth exposed to sunlight
To understand the influence of solar radiation on the DNA in
To evaluate teeth, the sample teeth were 51,
52, 27, 28, 29 and 30 of solar radiation
exposed. For this purpose, disposable plastic cups were again used
holes in the bottom for rainwater
to offer a drainage facility. So that
Teeth exposed to the sun without shade if possible
and to anchor the cups stably,
they were filled about two-thirds with small pebbles and on top the
Fig. 5: Location of in water
stored teeth
Fig. 6: Location of the solar radiation
exposed teeth
Sample teeth placed. The cups were then placed on a garage roof up
sunk into a gravel bed at the edge of the cup. So the teeth couldn't get out of the wind
be blown away and they were weather conditions, especially the
UV radiation from the sun, exposed (Fig. 6, p. 20).
Teeth were removed after about four weeks (teeth 51 and 52), four months
(27 and 28) and half a year (teeth 29 and 30) lay time.
2.3.5 Teeth made from dissected material
As already described in chapter 2.2.4, p. 18, there are two teeth (No. 33 and 34)
of sections in the Institute for Forensic Medicine in Würzburg.
The tooth of sample 33 was found on a heavily decayed corpse of a 36-year-old
Man, during which, according to the autopsy report, a lay time of around 17
Days is accepted. There were already blackish-green discolorations of the
Determine skin. The soft parts of the head and face were completely absent. More expansive
Maggot infestation could already be seen.
Sample 34 comes from the corpse of a 59-year-old man with the highest grade
Putrefaction and partly beginning mummification. The skin was discolored black.
In the area of ​​the midface, the skeleton was completely exposed and more massive
Maggot infestation was detectable. According to the autopsy report, the lay time was around 14
Days.
2.3.6 Teeth exposed to intense heat
The purpose of this experiment was to find out how much DNA in the tooth was under very hot conditions
Conditions such as those that can arise in a fire, for example, are degraded.
For this purpose, the teeth No. 11, 12, 13, 14, 16, 17, 18, 19 and 20 in a refractory
Glass containers in an oven preheated to 200 ° C in the Institute of Forensic Medicine
given.
After 30 min (teeth 11 and 12), 60 min (teeth 13 and 16), 90 min (teeth 17 and
18) and 120 min (teeth 19 and 20) the teeth were removed again and opened
Cooled to room temperature.
2.3.7 Teeth extracted 28 and 29 years ago
The teeth, which are from the maxillofacial surgery practice of Dr. Dr.
Hemmerich come from Munich, were 28 and 29 years ago
Years away. The storage of the H2O2
cleaned teeth were carried out dry in a glass container
at room temperature and without direct sunlight
(see Fig. 7).
2.3.8 Teeth from the excavation in Katzwang
As already described in chapter 2.2.3.1, p. 16,
these teeth come from an excavation in
Katzwang. To get as much tooth substance as possible
received, these teeth were contrary to the usual
The procedure is not circularly weakened and broken up,
but trepanned from the occlusal. To this too
Keeping the bore as small as possible was just that
large palatal root (all of these teeth were OK
Molars) and used for the examination (see Fig. 8).
2.3.9 Teeth from the Dietersberg cave
The single teeth of the excavation "Dietersberghöhle" (see Fig. 9) were only with
the system TPOX-vs and the Nonaplex-Multiplex, because of their age
little DNA was to be expected. Here, too, no tooth was weakened in a circular manner
and broken through, but like the teeth of the excavation in Katzwang
through holes from the palatal (sample teeth 48 and 49) or occlusal (sample tooth
50) opened.
Fig. 7: Storage vessel
of almost 30 years
old teeth
Fig. 8: Skull with sample tooth
No. 40 with trephination hole
2.4 Methods
2.4.1 Avoiding contamination
Contamination of the teeth as well as those that were removed
Samples containing foreign material must be avoided
to get a successful DNA analysis.
On the one hand, this means that foreign DNA can be included in the test series
get and give wrong results,
on the other hand it can contaminate the samples with
Bacteria or DNA-degrading enzymes for
Degradation of the DNA and thus false negatives
Lead to results. There is one during the entire preparation and processing
Contamination with foreign material possible. Sources of contamination to be mentioned in particular
In addition to the method for taking the sample, the laboratory staff,
the laboratory equipment, the pipettes, the pipette tips, the reaction vessels, the
Reagents, the centrifuge tubes, the thermal cycler and the electrophoresis chamber,
to name just a few examples (Newton et al. 1994, p. 53ff).
Various precautions were taken to avoid contamination
met. So only material from the inside
of the tooth was tried, became the teeth
both before creating a circular groove with a dental turbine
(see Fig. 10), as well as before breaking open the
Carefully clean the outside of the laboratory with one soaked in NaOCl
Cloth cleaned. NaOCl causes an oxidation of the
DNA remnants still adhering to the outside, causing this
denatured and thus destroyed. To cross contamination
to prevent with the previous sample,
were all instruments such as the lever for the leg, pliers, tweezers and endo files
Soaked in NaOCl after each use and then with a new one
Dried piece of paper roll (Gilbert et al. 2005; Yang et al. 2003).
Furthermore, a fresh piece was used as a base in the laboratory for each sample
Paper towel spread out. This did not have to be sterile, since the sample material with it
did not come into direct contact. The samples were strictly sterile
Fig. 10: Tooth with a circular
Groove in front of the opening of the pulp
Fig. 9: The three teeth from the
Dietersberg cave (samples 48-50)
Petri dish removed from the tooth. This was the case with every sample with new latex gloves
instead of.
All pipette tips that are not delivered sterile packed by the manufacturer
were sterilized in the autoclave before use.
To check for the presence of contaminated reagents ran out
Start with a neutral sample with each test series. In addition, one served
Negative control in the PCR for further security.
Additional precautions were taken as part of the preparation for the PCR
met. This created the space for making the master mix and distributing it
the PCR tubes are strictly separated from the place of addition of the DNA to a
To prevent "carry over" contamination.
Always wear fresh disposable gloves when pipetting the reagents
was obligatory.
After the result of the samples was available, it could also be compared with the known
genetic fingerprints of the author and the rest of the laboratory staff
another source of contamination can be excluded.
2.4.2 Sampling
The teeth were initially like before machining
described in chapter 2.4.1, p. 23, with a NaOCl
Soaked rag rubbed off to remove any adhering
Remove DNA residues. Subsequently were
the teeth in the dental practice Dr. Gebhard,
Schnaittach, with a diamond drill and one
Dental turbine at the level of the enamel-cement boundary
circularly weakened by the creation of a groove,
Sealed in shrink-wrap and only in the institute under
Laboratory conditions broken open, so that now the opened pulp and the root canals
were accessible. So there was contamination during transport
locked out.
Figure 11 shows the workplace in the Institute of Forensic Medicine, where the
Sampling from the opened teeth took place.
Fig. 11: Sampling location
With tooth extraction forceps and a small leg
Lever were the teeth on the previously applied
Predetermined breaking points broken (see Fig. 12). If
pulp tissue was still intact
this is removed with fine tweezers and placed in a
labeled centrifuge tube transferred. After that
the root canals became ascending with endo files
Size and the resulting dentine chips
collected in a petri dish.
2.4.3 DNA extraction
The samples obtained in this way were used to extract DNA using “E.Z.N.A.® Tissue
DNA Mini Kit “from PeqLab. This is a process in which
the DNA is selectively and reversibly bound to a silica membrane.
The procedure corresponds to the instructions enclosed with the kit. To do this will be
With the help of 200 μl TL buffer, transfer the chips from the Petri dish to a sterile 1.5 ml
centrifuge tube with a capacity of 25 μl of OBTM proteinase added through
Vortex carefully mixed and immersed in 55 ° C water for three hours.
Since no shaking water bath was available, we were also noisy
Instructions possible to mix the samples every 30 min by vortexing to avoid sedimentation
to prevent. For samples 1, 2, 7 to 10 and 26 to 35, the
Incubation carried out overnight in an incubator at 37 ° C, which is stated in the isolation protocol
is also indicated. This step is used to unlock the cell and
the breakdown of proteins.
Then 220 μl BL buffer is pipetted into the lysate by vortexing
mixed and incubated at 70 ° C in a water bath for ten minutes.
After adding 220 μl of absolute ethanol and vortexing, the entire batch is
loaded onto a HiBind® DNA column and centrifuged at 8,000 g for one minute.
The nucleic acids bind reversibly to the silica membrane of the HiBind® column.
The flow through is discarded.
By washing twice with 600 μl of DNA washing buffer each time, which is already
1.5 times the volume had been supplemented with absolute ethanol, and
Fig. 12: Sample tooth after the
Set out
Centrifugation for one minute at 8,000 g removes interfering PCR inhibitors.
The centrifugate is discarded in each case.
Now the membrane is at the maximum speed of the centrifuge (approx. 12,000 g)
completely dried for two minutes.
During the following elution, 200 μl of elution buffer preheated to 70 ° C. are added
the column matrix put into a fresh centrifuge tube is pipetted at room temperature
Incubated for three minutes and centrifuged at 8,000 g for one minute. Around
obtaining a higher concentration of DNA became the first for the second elution
Eluate also preheated to 70 ° C is used (PeqLab instructions).
2.4.4 Polymerase Chain Reaction (PCR)
PCR is an in vitro technique with which DNA sections can be amplified
can. All that is required is to know the start and end sequence of this section
be. The double-stranded DNA is melted at 94 ° C (denaturation) so that
it is present as a single strand. Oligonucleotide primers leading to the beginning and end sections
are complementary, attach to the DNA template. This
The process is called annealing and requires a temperature between 50
° C and 65 ° C depending on the primer. The DNA polymerase is stored in the 5’-direction from the primer
Deoxynucleoside triphosphates (dNTPs) corresponding to the single-stranded DNA template
at. This synthesis of the new strands of DNA is called extension and
takes place between 68 ° C and 72 ° C, depending on the polymerase. MgCl2 and a
added buffers are also essential for the PCR. MgCl2 promotes in one
Concentration between 1.0 mM and 1.5 mM the polymerase activity, but can be at
Too high a concentration has an inhibiting effect The buffer creates the right pH environment
for the reactions. The second begins with the renewed denaturation that now follows
Cycle. This leads to an exponential increase in the target sequence. The number of cycles
differs depending on the PCR kit used. Have passed through all cycles,
This is followed by a final 45-minute extension step in which
incompletely amplified DNA strands still need to be filled in. After all, it cools
Thermal cycler downs the samples to 4 ° C (Newton et al. 1994, p. 19ff).
A PCR reaction with 1 μl, 5 μl or 10 μl of DNA was set up for each sample
and with sterile distilled water. supplemented to a volume of 25 μl. The PCR was carried out in
the automatic thermal cyclers ABI Gene Amp PCR System 2400 (Applied Biosystems)
and Personal Cycler (Biometra). To activate DNA polymerase
the samples were heated to 94 ° C before the first PCR cycle.
2.4.4.1 STR SE33
Two primer sequences are used to amplify the STR single system SE33
required, which are composed as follows:
Primer 1: 5'-AATCTGGGCGACAAGAGTGA-3 '
Primer 2: 5’-ACATCTCCCCTACCGCTATA-3 ’
(Möller et al. 1994)
The following tables 6 and 7 give the composition of the master mix
and the conditions of the PCR again:
Reagent amount
Primer 1: 0.75 μl / batch
Primer 2: 0.75 μl / batch
dNTP: 1.00 μl / batch
10 x buffer: 2.50 μl / batch
MgCl2 2.00 μl / batch
Taq DNA polymerase: 0.20 μl / batch
Total: 7.20 μl / batch
Tab. 6: PCR approach for SE33
Phase temperature duration
Activation of Taq DNA polymerase: 94 ° C 10 min
Denaturation: 94 ° C for 1 min
Annealing: 60 ° C for 1 min
Extension: 72 ° C for 1 min
final extension: 60 ° C 45 min
Cooling: 4 ° C ¥
35 cycles
Tab. 7: PCR conditions for SE33
2.4.4.2 AmpFlSTR® SGM Plus Kit
The Multiplex AmpFlSTR® SGM Plus contains 11 x 2 fluorescence-marked primers,
through which the STR loci D3S1358, vWA, D19S539, D2S1338, D8S1179, D21S11,
D18S51, D19S433, THO1 and FGA, as well as the first intron sequence of the amelogenin
Gene can be amplified. With the help of this segment of the amelogenin gene is a
Gender differentiation possible (see Chapter 1.2.2.5, p. 6).
The systems of the AmpFlSTR® SGM Plus Kit are listed in Table 8:
Chromosomal locus
place
Repetitive motif size in
bp
Fluorescent marking
D3S1358 3p TCTA [TCTG] 1-2 [TCTA] n 114-142 5-FAM
vWA 12p12-pter TCTA [TCTG] 3-4 [TCTA] n 157-209 5-FAM
D16S539 16q24-qter [AGAT] n 234-274 5-FAM
D2S1338 2q35-37.1 [TGCC] n [TTCC] n 289-341 5-FAM
Amelogenin X: p22.1-22.3 107 JOE
Amelogenin Y: p11.2 113 JOE
D8S1179 8 [TATR] n; R can be A or C 128-172 JOE
D21S11 21q11.2-q21
[TCTA] n [TCTG] n [TCTA] 3 TA [TCTA] 3 TCA
[TCTA] 2 TCCATA [TCTA] n
187-243 JOE
D18S51 18q21.3 [AGAA] n 26-345 JOE
D19S433 19q12-13.1 [AAGG] [AAAG] [AAGG] [TAGG] [AAGG] n 106-140 NED
THO1 11p15.5 defaultn 165-204 NED
FGA 4q28 [TTTC] 3 TTTTTTCT [CTTT] n CTCC [TTCC] 2 215-353 NED
Tab. 8: Systems of the AmpFlSTR® SGM Plus® (according to the instructions for the AmpFlSTR® SGM Plus®, Applied
Biosystems)
The AmpFlSTR® SGM Plus Kit already contains the AmpFlSTR® PCR Reaction Mix,
the AmpFlSTR® Profiler Plus Primer Set and the AmpliTaq Gold® DNA Polymerase
(5 U / µl). The AmpFlSTR® PCR Reaction Mix is ​​a mixture of a dNTP mix,
Mg2 + and BSA (Bovine serum albumin). The added BSA can be PCR-inhibiting
Minimize influences by binding the inhibitors (Comey et al. 1994).
The two following tables 9 and 10 give the composition of the master mix
and the conditions prevailing in the PCR again:
Reagent amount
ABI AmpFlSTR® Profiler Plus Primer Set: 2.50 μl / approach
ABI AmpFlSTR® PCR Reaction Mix: 5.00 μl / batch
AmpliTaq Gold® DNA Polymerase: 0.50 μl / batch
Total: 7.50 μl / batch
Tab. 9: PCR approach for AmpFlSTR® SGM Plus® (according to the AmpFlSTR® SGM Plus® instructions, Applied
Biosystems)
Phase temperature duration
Activation of the AmpliTaq GoldTM DNA polymerase: 94 ° C 1 min
Denaturation: 94 ° C for 1 min
Annealing: 59 ° C for 1 min
Extension: 72 ° C for 1 min
final extension: 60 ° C 45 min
Cooling: 4 ° C ¥
28 cycles
Tab. 10: PCR conditions for AmpFlSTR® SGM Plus® (according to the AmpFlSTR® SGM
Plus®, Applied Biosystems)
2.4.4.3 STR TPOX-vs
Like the STR system SE33 (see chapter 2.4.4.1; p. 27) the STR system is TPOXvs
a single system determined by the following two primer sequences
becomes:
Primer 1: 5'-CCTGTTCCTCCCTTATTTCC-3 '
Primer 2: 5'-GAACACAGACTCCATGGTG-3 '
New primers shorten the section to be amplified from 218-246 bp
(TPOX) to 58-86 bp (TPOX-vs) (Hellmann et al. 2001).
The master mix for the PCR approaches is made up as listed in Table 11
together. Table 12 shows the program for amplification in the thermal cycler:
Reagent amount
Primer 1: 0.75 μl / batch
Primer 2: 0.75 μl / batch
dNTP: 1.00 μl / batch
10 x buffer: 2.50 μl / batch
MgCl2 2.00 μl / batch
Taq DNA polymerase: 0.20 μl / batch
Total: 7.20 μl / batch
Tab. 11: PCR approach for TPOX-vs (Hellmann et al. 2001)
Phase temperature duration
Activation of Taq DNA polymerase: 94 ° C 10 min
Denaturation: 94 ° C for 1 min
Annealing: 55 ° C for 1 min
Extension: 72 ° C for 1 min
final extension: 60 ° C 45 min
Cooling: 4 ° C ¥
35 cycles
Tab. 12: PCR conditions for TPOX-vs (Hellmann et al. 2001)
2.4.4.4 Mentype® NonaplexQS PCR Amplification Kit
The Mentype® NonaplexQS PCR Amplification Kit contains the Loci D3S1358,
D8S1179, D18S51, D21S11, FGA, SE33, THO1 and vWA. In addition, in turn
amplified the first intron sequence of amelogenin.
Table 13 gives an overview of the systems used in the Mentype® NonaplexQS:
Locus
Chromosomal
Place Repetitive motif
Reference allele
Size in
bp
Fluorescent marking
Amelogenin Xp22.1-22.3 83 6-FAM
Amelogenin Yp11.2 87 6-FAM
D8S1179 8q23.1-23.2 [TATC] 12 12 95-163 6-FAM
D21S11 21q21.1
[TCTA] 4 [TCTG] 6 [TCTA] 3 TA [TCTA] 3 TCA [TCTA] 2
TCCATA [TCTA] 11
29 177-252 6-FAM
D18S51 18q11.1 [AGAA] 13 13 281-427 6-FAM
THO1 11p15.5 default9 9 82-131 HEX
D3S1358 3p25.3 TCTA [TCTG] 2 [TCTA] 15 18 139-195.5 HEX
SE33 6q14.2 [AAAG] 9 AA [AAAG] 16 25.2 196-377 HEX
vWA 12p13.31 TCTA [TCTG] 4 [TCTA] 13 18 102-168 NED
FGA 4q28.2 [TTTC] 3 TTTTTTCT [CTTT] 13 CTCC [TTCC] 2 21 174-338 NED
Tab. 13: Mentype® NonaplexQS systems (according to instructions for Mentype® NonaplexQS, Biotype
® and homepage Biotype AG)
The scope of delivery of this kit contains a reaction mixture (Reactions Mix A),
which already contains Mg2 +, dNTP mix and BSA, and a primer mix (primer
Mix). In addition, JumpStartTM Taq DNA Polymerase (Sigma; 2.5 U / μl)
required, which must be activated for one minute at 94 ° C before the PCR.
The following two tables show the composition of the master mix for the
PCR approach and the PCR process:
Reagent amount
Primer Mix: 2.50 μl / batch
Reaction Mix A: 5.00 μl / batch
JumpStartTM Taq DNA Polymerase: 0.40 μl / batch
Total: 7.40 μl / batch
Tab. 14: PCR approach for Mentype® NonaplexQS (according to instructions for Mentype® NonaplexQS, Biotype
®)
Phase temperature duration
Activation of JumpStartTM Taq DNA Polymerase: 94 ° C 1 min
Denaturation: 94 ° C for 1 min
Annealing: 60 ° C for 1 min
Extension: 72 ° C for 1 min
final extension: 68 ° C 45 min
Cooling: 4 ° C ¥
35 cycles
Tab. 15: PCR conditions for Mentype® NonaplexQS (according to instructions for Mentype® NonaplexQS,
Biotype®)
2.4.5 Gel electrophoresis and silver staining
In gel electrophoresis, proteins,
Nucleic acids or other charged macromolecules
in the electric field according to their size, shape and
Disconnect net charge. For the separation of DNA molecules
less than 500 nucleotides is particularly suitable
PAGE (polyacrylamide gel electrophoresis),
because, contrary to the agarose gel, they have very small pores
having. Since the DNA carries a negative charge, it migrates
them in an electric field to the anode. As
The result is bands of people who have migrated equally far
Molecules (see Fig. 13) (Kreutzig 2002, p.
117).
After the electrophoresis was completed, the
Electrode strips removed and the gel in a glass dish
given. The gel was used for equilibration
1% nitric acid (HNO3) was added and it
was on a tumble shaker for five minutes
posed. Then the gel became about ten
Fig. 13: Polyacrylamide gel on the
Cooling plate
Fig. 14: Stained foreleg of the
Samples 51 and 52; left: the SE33,
right: SGM multiplex; both with
Conductor, neutral, positive and
Negative control
Seconds with distilled water. rinsed and with freshly made silver nitrate solution for
Incubated on the shaker for twelve minutes. After rinsing thoroughly again with
Aqua dest. the developer solution was added and until the
PCR bands awaited. A 10% -
ige vinegar solution, which had to act for at least two minutes. To the gels
To make them storable, they were treated with distilled water. rinsed thoroughly for five minutes
placed in 5% glycerol and finally after one night at 80 ° C in the heating cabinet
covered with foil (see Fig. 14, p. 31) (Allen et al. 1989).
2.4.6 High resolution capillary electrophoresis
With the help of capillary electrophoretic separation (GeneScan) in the automatic
Automatic sequencing Genetic Analyzer ABI 310 (Applied Biosystems) is
possible to analyze the STR systems. In a capillary filled with polymer
electrophoresis takes place here (Ziegle et al. 1992).
It is possible to determine the length of the fragments down to one base pair.
After laser-assisted fragment length measurement, this is confirmed by a comparison with
an internal length standard (GeneScan 500 ROX Size Standard, Applied
Biosystems). The fluorescent markers are used for differentiation
overlapping systems (Kimpton et al. 1993). An allelic ladder separated in parallel
is used to assign the fragment lengths to a defined allele.
The following table shows the corresponding quantities of the reagents
again:
Reagent amount
Formamide: 12.00 μl / batch
GeneScan 500 ROX Size Standard: 0.50 μl / batch
Total: 12.50 μl / batch
Tab. 16: Approach for capillary electrophoresis
After the master mix was distributed to the sample vessels, that became
analyzing the PCR product depending on the intensity of the bands
PAGE gel (between 0.5 and 3.0 μl) added. For linearizing the DNA
the batches were incubated at 95 ° C. for 150 seconds in the heating block and
then analyzed in the sequencing machine.
2.4.7 Quantifiler ® Human DNA Quantification Kit
Using the Quantifiler® Human DNA Quantification Kit and the PRISM® 7000
Sequence Detection System it is possible to determine DNA concentrations.
A real-time quantitative PCR (RTQ-PCR) is used to produce a 62 bp
Intron of the human telomerase reverse transcriptase gene (hTERT) amplified. These
Target is at 5p15.33. The Quantifiler® Human Primer Mix consists
from the two primers with fluorescent labeling (FAM). In the Quantifiler® PCR
Reaction Mix are already the AmpliTaq Gold® DNA polymerase, dNTPs and a
Buffers included. In addition, a dilution series from eight standards is required
can be made, which is used for later comparisons. For this purpose, 10 μl des
Quantifiler Human DNA Standards (200 ng / μl) with 30 μl T10E0.1 (10 mM Tris-HCl [pH
8.0], 0.1 mM Na2EDTA). The standard 1 (50 ng / μl) is again 10 μl
mixed with 30μl T10E0.1 up to a concentration of 0.023 ng / μl for standard 8
is present.
The table shows the composition of the master mix that was added to the 2 μl of DNA
must become.
Reagent amount
Quantifiler® Human Primer Mix: 10.50 μl / batch
Quantifiler® PCR Reaction Mix: 12.50 μl / batch
Total: 23.00 μl / batch
Tab. 17: Preparation for Quantifiler® Human DNA Quantification Kit (according to the Quantifiler® Human
DNA Quantification Kit, Applied Biosystems)
With the RTQ-PCR the samples are determined by fluorescence measurements in the range of
500-660 nm analyzed and evaluated by comparison with the standards
(Instructions for Quantifiler® Human DNA Quantification Kit, Applied Biosystems).
The accompanying neutral sample can be used to check whether devices and
Reagents were DNA free.


3 results
The sample consisted of 50 teeth. These were different storage conditions
suspended and checked whether a DNA analysis is then carried out
could or whether too strong degrading influences have already acted.
After the DNA extraction, the approaches were initially carried out using the SE33
and the SGM-Multiplex-Kit amplified and evaluated. Could in the evaluation
no clear typing can be made, the samples were with further
Kits amplified. In cases in which the STR system SE33 could not be detected,
an attempt was made with the help of the STR TPOX-vs, which only contains very short DNA fragments
required for analysis to obtain a typification. How to determine if
amplifiable DNA is present at all. If typing with the STRSystem
SE33 was successful, but with the SGM the result was not satisfactory
failed, attempts were made to do DNA analysis with the more sensitive Nonaplex
Get exams. Likewise, was detectable
TPOX-vs then amplifies the DNA with the Nonaplex kit.
Finally, all DNA samples were collected using the Quantifiler® Human DNA
Quantification Kits (Applied Biosystems) subjected to quantification.
The result was assumed to be successfully typable if the peak was at
the GeneScan evaluation sufficiently large (greater than 30 rfu [relative fluorescent
units]) on an allele range. All other peaks, which are only
slightly raised from the noise (less than 30 rfu), but still
were clearly on an allele range, were indicated by a question mark "(?)"
made (see Fig. 15, p. 35). If no allele could be determined, it would
marked by "***".
The sample number was assigned according to the sequence of the machined teeth.
3.1 Freshly extracted teeth
Samples 1 to 5 come from teeth that were used before the tooth extraction
had reacted sensitively to a cold test. Your tooth pulps were thus to
Time of removal still vital. With the systems SE33 and SGM-Multiplex
these teeth were successfully detectable. The DNA concentrations obtained
were between 0.374 and 15.4 ng / μl.
The tooth of sample 6 no longer reacted before the tooth extraction was DNA sensitive
no longer detectable with the quantifiler. The systems SE33 and the SGMMultiplex
gave no amplifiable result. The TPOX-vs system delivered
Fig. 15: Upper graphic: The reference ladder of this system is used for comparison with the
Sample results. Middle graphic: A result that cannot be clearly detected. The two peaks
of sample 26 are in the range of alleles 16 and 17, but only appear very weak
Background noise and are therefore marked with a “?”. Lower graphic: At
Sample 47 a clear allele assignment is possible.
just like the nonaplex multiplex, a complete, clearly detectable one
Feature pattern, only the locus D8S1179 was classified as questionable.
Samples 7 to 9 are material
of deeply carious teeth (see Fig. 16). The tooth decay
was already so advanced that large parts
the crown was missing. A probe couldn’t
direct connection to the pulp can be established because
the tooth pulp is already withdrawn at this point
and had formed irritable dentine. Tried with that
the body to defend itself against the invading microorganisms. in the
Tooth, only reversible pulpitis is produced, which is not degrading
Has properties. The systems SE33 and the nonaplex multiplex could
be typed in all loci. The DNA concentrations were between 0.092 and
0.335 ng / µL.
Sample 14 was taken from a tooth in which the pulp cavity is strong
was obliterated and the canals can only be found and processed with difficulty
could. With very fine endo needles of ISO size 10 (0.1 mm diameter
the tip) it was possible to find the channel and eventually up to ISO size 30
to prepare. Only the STR system SE33 and the Locus TH01 of the
Nonaplex-Multiplex show a result. The SGM multiplex analysis was like that
Determination of concentration, unsuccessful.
Sample 15, which was obtained from a wisdom tooth, had root growth
not yet fully completed. Despite wide open apical
Foramina could easily obtain a DNA analysis using SGM and SE33
become. The DNA concentration was 11.42 ng / μl.
In addition, two teeth with root canal fillings (samples 24 and 25)
be examined (see Chapter 1.3, p. 8). The amount of DNA was <5 pg / μl
below the detection limit, but sample 24 (with a one year
old root canal filling) the SE33 system and five loci of the nonaplex multiplex
(FGA, TH01, D3S1358, vWA and AMEL) can be successfully amplified. The analysis
of the multiplex SGM was not successful in any system. The tooth of sample 25 had
a six year old root canal filling. The SE33 system was unsuccessful,
whereas the system TPOX-vs and a locus of the nonaplex multiplex (TH01)
Brought success. With two loci of the SGM (D18S51 and vWA) and one locus of the
Fig. 16: Deeply destroyed tooth of the
Sample No. 9
Nonaplex multiplex (D18S51) alleles could be suspected. However, it was
Locus D18S51 in the two kits, AmpFl STR® SGM Plus and NonaplexQS PCR
Amplification, mismatched. The Quantifiler® Human DNA Quantification Kit
could not detect any DNA (see Tab. 18 (a) and 18 (b)).
Sample amount in
No. ng / μl FGA D19S433 D18S51 TH01 D8S1179 D21S11 D3S1358 vWA D16S539 D2S1338 AMEL
1 15.4 26.2 / 27.2 21/22 13/14 15/17 7/9 15/16 28/29 16 17/18 11 24/25 XY
2 0.374 25.2 / 26.2 21/23 14 / 15.2 14/20 5 / 9.3 10/16 28/30 16/18 14/17 12/13 22/24 XY
3 5.21 14 / 21.2 19/25 15 / 15.2 14 7 / 9.3 12/13 29 / 31.2 16/18 14/16 11/12 17/19 XY
4 7.26 15/17 21 13 15/17 6/7 14/15 27 / 33.2 17 17 11/12 20/23 XY
5 7.11 15/17 21 13 15/17 6/7 14/15 27 / 33.2 17 17 11/12 20/23 XY
6 <5pg *** *** *** *** *** *** *** *** *** *** *** *** X
7 0.126 20 / 20.2 20/22 14/15 14/17 9.3 13/14 30 / 31.2 15/16 14/17 9/11 21/24 X
8 0.335 19 / 29.2 20/27 13/14 16/19 8 / 9.3 14 30 / 31.2 14/15 17/18 9/12 23/24 XY
9 0.092 19 / 29.2 20/27 13/14 16/19 8 / 9.3 14 30 / 31.2 14/15 17/18 9/12 23/24 XY
14 <5pg 17.2 *** *** *** *** *** *** *** *** *** *** ***
15 11.42 28.2 / 29.2 19/21 13/15 12/17 6/9 13/14 28/30 14/16 16/18 12 18/24 X
24 <5pg 16 / 23.2 *** *** *** *** *** *** *** *** *** *** ***
25 <5pg *** *** *** 10.2 (?) *** *** *** *** 15.2 (?) *** *** ***
SE33 SGM
Table 18 (a): Quantification, SE33 and SGM of the freshly extracted teeth
Sample no.
SE33 FGA D18S51 TH01 D8S1179 D21S11 D3S1358 vWA AMEL
1 no Nonaplex and TPOX-vs, since SGM was successful fresh
2 no Nonaplex and TPOX-vs, since SGM was successful fresh; vital
3 no Nonaplex and TPOX-vs, since SGM was successful fresh; vital
4 no Nonaplex and TPOX-vs, since SGM was successful fresh; vital
5 no Nonaplex and TPOX-vs, since SGM was successful fresh; vital
6 8/9 15 / 28.2 20/25 12/20 9.3 12/14 (?) 28 / 31.2 16 16/17 X fresh; avital
7 no Nonaplex and TPOX-vs, since SGM was successful fresh; deeply destroyed
8 no Nonaplex and TPOX-vs, since SGM was successful fresh, deeply destroyed
9 no Nonaplex and TPOX-vs, since SGM was successful fresh, deeply destroyed
14 SE33 successful *** *** *** 9 *** *** *** *** *** fresh; 83J; severely sclerosed
15 no Nonaplex and TPOX-vs, since SGM was successful fresh; wide open F.a.
24 SE33 successful *** 20 *** 8/10 *** *** 17 16 XY fresh; 1 year old WF
25 8 *** *** 13 (?) 8 / 9.3 *** *** *** *** *** fresh; 6 year old WF
TPOX vs. Nonaplex comment
Table 18 (b): TPOX-vs and Nonaplex of the freshly extracted teeth
3.2 Teeth buried in the ground
Sample 10, which was buried in the ground for four weeks, produced a complete one
Feature pattern in the STR system SE33 and the SGM multiplex. The amount of
DNA of 0.019 ng / μl was sufficient for this.
After three months (sample 26) there was already a slight degradation of the DNA
occurred. In contrast to the SE33 system, the SGM multiplex delivered
incomplete feature pattern. The systems D19S433, D3S1358 and AMEL
could be clearly typed for the loci FGA, TH01, D8S1179, D21S11,
vWA, D16S539 and D2S1338 the peaks were only very weak. The
Locus D18S51 gave no usable result. The potential of the SGM multiplex
recognizable loci could be completely confirmed in the nonaplex multiplex.
Only in the FGA locus was only one allele suspected in the SGM multiplex and in the
Two alleles were detected in the nonaplex multiplex. The existing concentration of DNA
was 0.042 ng / µL.
Samples 31 and 32 are from teeth that were buried for about half a year
were. The STR SE33 was successful with both, but the SGM multiplex
Approach no longer sufficient for a complete feature pattern. Only at
sample 31 could have four loci (D19S433, D21S11, D16S539 and AMEL) and at
of sample 32 eight loci (FGA, D18S51, D8S1179, D21S11, D3S1358, vWA,
D16S539 and AMEL) of the SGM multiplex can be partially detected. The DNA analytical
Investigations resulted in sample 31 at the SGM multiplex locus D21S11
an allele pattern with two peaks, only one of which is confirmed in the nonaplex
could. The SGM multiplex also produced an incorrect result for sample 31
Gender; the allele for "Y" was not amplified. Locus D8S1179 of sample 31
could not be determined using nonaplex multiplex either.
After about a year, the trial teeth 43, 44 and 45 were out of the ground
taken. Only a few alleles could be identified in these samples
become. Only the very short TPOX-vs and the AMEL locus of the Nonaplex
could still be successfully amplified in all three. At sample 43 it was
in addition, locus TH01 of the nonaplex multiplex was successful, at sample 44 the
Locus SE33 of the nonaplex multiplex and for sample 45 the loci TH01 and D8S1179
of the nonaplex multiplex. The amount of DNA in all was below the detection limit
of 5 pg / μl.
Sample amount in
No. ng / μl FGA D19S433 D18S51 TH01 D8S1179 D21S11 D3S1358 vWA D16S539 D2S1338 AMEL
10 0.019 15 / 30.2 23/25 13/15 12/19 7 / 9.3 10/13 28/30 18 14/16 11 18/23 X
26 0.042 20 / 30.2 22 (?) 13 / 15.2 *** 7/9 (?) 11/13 (?) 28 / 32.2 (?) 17/18 16/17 (?) 12 / 13 (?) 23 (?) X
31 <5pg 19 *** 14 (?) *** *** *** 19 / 34.2 (?) *** *** 9 *** X
32 0.051 14/19 25 (?) *** 14 *** 14 29 15/16 16/17 11/12 (?) *** XY
43 <5pg *** *** *** *** *** *** *** *** *** *** *** *** ***
44 <5pg 17 *** *** *** *** *** *** *** *** *** *** ***
45 <10pg *** *** *** *** *** *** *** *** *** *** *** *** ***
SE33 SGM
Tab. 19 (a): Quantification, SE33 and SGM of the buried teeth
Sample no.
SE33 FGA D18S51 TH01 D8S1179 D21S11 D3S1358 vWA AMEL
10 no Nonaplex and TPOX-vs, since SGM was successful Earth: July 9th-August 15th, 2005
26 SE33 successful 20 / 30.2 18/22 15/17 7/9 11/13 28 / 32.2 17/18 16/17 X Earth: 09.07.-15.10.05
31 SE33 successful 19 19 19 6 *** 34.2 15/17 *** XY Earth: 09.07.05-03.03.06
32 SE33 successful 14/19 25 14 7/10 14 29 15/16 16/17 XY Earth: 09.07.05-03.01.06; vital
43 8/11 *** *** *** 6 *** *** *** *** X Earth: 07/09/05- 07/24/06; vital
44 11 *** *** *** *** *** *** *** *** X Earth: 7/9/05 - 7/24/06; vital
45 8/11 *** *** *** 6 13/14 *** *** 15 XY Earth: 07.09.05-24.07.06; vital
TPOX vs. Nonaplex comment
Table 19 (b): TPOX-vs and Nonaplex of the buried teeth
3.3 Teeth stored in water
During the analysis, from the sample that had been stored in the water for four weeks, only 35 could
0.078 ng / μl DNA can be obtained. Admittedly, this was sufficient to use the system
SE33 and the nonaplex multiplex to achieve a DNA analysis, however, could
the SGM multiplex cannot be clearly typed at all loci. The five successful
amplified and the three suspected loci of the SGM multiplex agreed
those of the nonaplex multiplex, but the loci D8S1179, D16S539
and D2S1338 cannot be reproduced in the SGM multiplex.
Samples 42 (0.511 ng / μl DNA) and 43, which had been in water for half a year
(0.666 ng / μl DNA) were with the single system SE33 and the SGM multiplex
typeable, so that the PCR can neither be performed with the TPOX-vs nor with the
Nonaplex multiplex had to be carried out.
After a year the results were less successful. The quantification kit
could no longer detect any DNA. For sample 36, the systems were SE33 and
TPOX-vs successful, but both with SGM multiplex and with nonaplex
Multiplex only appeared about half of the systems. Were successful
in the SGM multiplex the loci FGA, TH01, D8S1179, D21S11, D3S1358, vWA and
AMEL, with the nonaplex multiplex the loci D18S51, TH01, D21S11, D3S1358, vWA
and AMEL. Sample 37 was neither with the SE33 system nor the SGM multiplex
Typing is possible, but the TPOX-vs system successfully delivered a
typed result. The nonaplex multiplex also resulted in loci TH01, D21S11,
D3S1358, vWA and AMEL an exploitable success.
Sample amount in
No. ng / μl FGA D19S433 D18S51 TH01 D8S1179 D21S11 D3S1358 vWA D16S539 D2S1338 AMEL
35 0.078 23.2 / 26.2 20/22 15 (?) 12/16 7/9 *** 28/30 (?) 15/16 (?) 15/17 *** *** XY
41 0.511 13 / 32.2 24/25 13/14 12/15 7 / 9.3 13/14 30/31 16/17 15/18 11/12 18 X
42 0.666 18 / 30.2 21/26 13/15 12 6/10 13 27/30 15/17 14 13 20 X
36 <5pg 17 / 29.2 17/18/19/24 *** *** 6/9 14 32.2 / 34.2 15 15 *** *** XY
37 <5pg *** *** *** *** *** *** *** *** *** *** *** ***
SE33 SGM
Tab. 20 (a): Quantification, SE33 and SGM of the teeth stored in water
Sample no.
SE33 FGA D18S51 TH01 D8S1179 D21S11 D3S1358 vWA AMEL
35 SE33 successful 23.2 / 26.2 20/22 12/16 7/9 12/15 28/30 15/16 15/17 XY H2O: 23.09.05-03.01.06
41 no Nonaplex and TPOX-vs, since SGM was successful H2O: 23.09.05-03.03.06; vital
42 no Nonaplex and TPOX-vs, since SGM was successful H2O: 23.09.05-03.03.06; vital
36 8 *** *** 15 (?) 6/9 *** 31 16/18 14/15 XY H2O: 23.09.05-24.09.06; vital
37 9 *** *** *** 9 (?) *** 29 14 (?) 15/16 X H2O: 23.09.05-24.09.06
TPOX vs. Nonaplex comment
Tab. 20 (b): TPOX-vs and Nonaplex of the teeth stored in water
3.4 Teeth exposed to sunlight
Samples Nos. 51 and 52 gave after four weeks of exposure to sunlight
System TPOX-vs and with the nonaplex multiplex a clear typing. The
DNA concentrations of 0.823 and 0.947 ng / μl were sufficient for this.
After three months, no more DNA could be found in samples 27 and 28
be quantified. The PCR using the STR system SE33 was also unsuccessful
and the SGM multiplex. Only the TPOX-vs system could be typed. The
Nonaplex multiplex produced three typable loci in sample 27 (TH01, D3S1358
and AMEL) and in sample 28 only one typable locus (TH01) emerged.
After half a year, the sample of tooth # 29 with no kit showed one
Typing. The quantification could not detect any DNA either. The sample 30
could only be typed in the TPOX-vs system. The two loci in the nonaplex multiplex, which
only showed very faint peaks, cannot be unambiguously identified
can be used.
Sample amount in
No. ng / μl FGA D19S433 D18S51 TH01 D8S1179 D21S11 D3S1358 vWA D16S539 D2S1338 AMEL
51 0.823 no SE33 and SGM, as TPOX-vs and Nonaplex were made
52 0.947 no SE33 and SGM, as TPOX-vs and Nonaplex were made
27 <5pg *** *** *** *** *** *** *** *** *** *** *** *** ***
28 <5pg *** *** *** *** *** *** *** *** *** *** *** *** ***
29 <5pg *** *** *** *** *** *** *** *** *** *** *** *** ***
30 <5pg *** *** *** *** *** *** *** *** *** *** *** *** ***
SE33 SGM
Tab. 21 (a): Quantification, SE33 and SGM teeth exposed to solar radiation
Sample no.
SE33 FGA D18S51 TH01 D8S1179 D21S11 D3S1358 vWA AMEL
51 8 14/19 20/24 17 6 ​​/ 9.3 12/13 28 / 31.2 16/17 17 X UV: 27.12.06-01.02.06
52 10/11 18 / 30.2 21/26 12 6/10 13 27/30 15/17 14 X UV: 27.12.06-01.02.06
27 9 *** *** *** 9.3 *** *** 17 *** XY UV: 04.08.-05.11.05; vital
28 8/9 *** *** *** 9.3 *** *** *** *** *** UV: 04.08.-05.11.05
29 *** *** *** *** *** *** *** *** *** *** UV: 04.08.05-03.03.06; vital
30 8/11 *** *** *** 6 *** *** *** *** X (?) UV: 04.08.05-03.03.06
TPOX vs. Nonaplex comment
Tab. 21 (b): TPOX-vs and Nonaplex teeth exposed to sunlight
3.5 teeth made of dissected material
With sample 33 (about 17 days in place) 0.091 ng / μl DNA could be obtained.
A DNA-analytical examination using the SE33 system and the SGMMultiplex
was possible in all systems.
Sample 34 (about 14 days left) no longer provided any quantifiable DNA. The
The result of the examination with the STR system SE33 was successful, with the SGMMultiplex
could not do three loci (D18S51, TH01 and D2S1338) and only five others
uncertainly (FGA, D19S433, D21S11, vWA and D16S539).
Only with the loci D8S1179, D3S1358 and AMEL could the alleles be unambiguous
be determined.
SGM
FGA D19S433 D18S51 TH01 D8S1179 D21S11 D3S1358 vWA D16S539 D2S1338 AMEL
33 0.091 27.2 / 33.2 20/23 15.2 13/14 7/8 11/13 29/30 14 17 11/12 18/25 XY
34 <5pg 18 21/22 (?) 16 (?) *** *** 11/14 28 (?) 17 19 (?) 12 (?) *** XY
Samples- SE33
No
Amount in
ng / μl
Tab. 22 (a): Quantification, SE33 and SGM of the teeth from the section material
SE33 FGA D18S51 TH01 D8S1179 D21S11 D3S1358 vWA AMEL
33 SE33 successful 27.2 / 26.2 20/23 13/14 7/8 11/13 29/30 14 17 XY decomposed corpse ~ 17d
34 SE33 successful *** *** *** 7/9 *** *** *** *** *** decomposed corpse ~ 14d
Samples- TPOX- vs. Nonaplex Comment
No
Tab. 22 (b): TPOX-vs and Nonaplex of the teeth from the section material
3.6 Teeth exposed to intense heat
The DNA concentration decreased continuously with increasing exposure to heat.
After 30 minutes, concentrations of 0.154 ng / μl (sample 11) and 1.79 ng / μl were still
(Sample 12), values ​​of <10 pg / μl (sample
13) and 0.034 ng / μl (sample 16) can be determined. All values ​​from later samples (No. 17 -
20) were below the detection limit.
Corresponding to this is the detection of the various alleles. At
The SE33 system was able to reliably detect the first samples (No. 11 and 12)
and also in the SGM multiplex there were four (sample 11:
the loci FGA, D19S433, D21S11 and AMEL) or five (sample 12: the loci D19S433,
D8S1179, D21S11, vWA and AMEL) amplifiable loci. The nonaplex multiplex
only gave no result for sample 11 in system D21S11.
Samples 13 and 16, taken after 60 minutes, also brought in
STR system SE33 one result. With the SGM multiplex, however, could only with
Sample 16 of the AMEL locus can be reproduced, but not with the
Gender of the patient matched. With the nonaplex multiplex it was possible
more loci are typed (sample 13: SE33, TH01, and AMEL; sample 16: FGA,
TH01, D21S11, vWA and AMEL), but this is enough for a satisfactory
Identification does not matter.
90 minutes after the start of the experiment, samples no. 17 and 18 were placed in the oven
taken. Sample 17 could still be typed in the SE33 system. This succeeded
at sample 18 no more. The SGM multiplex kit enabled both samples
no typing. Another examination of sample 18 with the system
TPOX-vs could represent alleles. Examination with the Nonaplex kit allowed
A typing of the AMEL locus for both samples and additionally for sample 18
the loci TH01 and D3S1358.
Teeth # 19 and # 20 were exposed to heat for 120 minutes. Sample 19 could
can still be typed in the SE33 system, sample 20 did not give any result. First
Typing was achieved with the aid of the TPOX-vs system. The SGMMultiplex
could not provide any alleles in either. The nonaplex multiplex supplied
both alleles usable at the D21S11 locus, and also at the TH01 locus
represents an allele in sample 20.
Sample amount in
No. ng / μl FGA D19S433 D18S51 TH01 D8S1179 D21S11 D3S1358 vWA D16S539 D2S1338 AMEL
11 0.154 19 / 27.2 18.3 13/14 *** *** *** 32.2 *** *** *** *** Y
12 1.79 18/20 *** 14 *** *** 11/12 29/30 17/18 16/18 *** *** XY
13 <10pg 18 *** *** *** *** *** *** *** *** *** *** *** ***
16 0.034 27.2 *** *** *** *** *** *** *** *** *** *** *** X
17 <5pg 32 *** *** *** *** *** *** *** *** *** *** *** ***
18 <5pg *** *** *** *** *** *** *** *** *** *** *** *** ***
19 <5pg 20 / 28.2 *** *** *** *** *** *** *** *** *** *** ***
20 <5pg *** *** *** *** *** *** *** *** *** *** *** *** ***
SE33 SGM
Tab. 23 (a): Quantification, SE33 and SGM of teeth exposed to strong heat
Sample no.
SE33 FGA D18S51 TH01 D8S1179 D21S11 D3S1358 vWA AMEL
11 SE33 successful 27.2 / 29.2 16/21 17 8 / 9.3 14 *** 14/17 16/17 XY oven: 30min / 200 ° C; deeply destroyed
12 SE33 successful 18/20 20/25 16 8 / 9.3 11/12 29/30 17/18 16/18 XY furnace: 30min / 200 ° C
13 SE33 successful 28.2 *** *** 6/8 *** *** *** *** XY furnace: 60min / 200 ° C
16 SE33 successful *** 18 *** 7 *** 27 / 36.2 *** 14 XY furnace: 60min / 200 ° C
17 SE33 successful *** *** *** *** *** *** *** *** XY (?) Oven: 90min / 200 ° C
18 8/11 *** *** *** 9 *** *** 17/18 *** XY furnace: 90min / 200 ° C
19 SE33 successful *** *** *** *** *** 34 *** *** *** Oven: 120min / 200 ° C
20 8/9 *** *** *** 6 *** 27 / 32.2 *** *** *** furnace. 120min / 200 ° C
TPOX vs. Nonaplex comment
Tab. 23 (b): TPOX-vs and Nonaplex of teeth exposed to intense heat
3.7 Teeth extracted 28 and 29 years ago