EAMHMS

SYMPOSIUM AND WORKSHOP

2015

NONDESTRUCTIVE DNA TECHNIQUES

IN HISTORICAL MATERIALS

WROCŁAW (POLAND) SEPTEMBER 24-26.2015

The Workshop is held under the honorary auspices of Polish Ministry of Culture and National Heritage and His magnificence Chancellor of the Wrocław Medical University

Organizers

European Association of Museums of the History of the Medical Sciences (EAMHMS)

Department of Forensic Medicine Wrocław Medical University

Institute of Molecular Techniques

Department of Humanistic Sciences in Medicine Wrocław Medical University

Oraganizing Commitee

Prof. Tadeusz Dobosz (Chair)

Willem J. Mulder (Coordinator)

Beata Bartnik

Grażyna Dmochowska

Anna Jonkisz

Anna Karpiewska

Elżbieta Kowalczyk

Arleta Lebioda

Małgorzata Małodobra-Mazur

Miron Tokarski (Conference Officer)

Mariola Turkiewicz

Day 1 - Thursday September 24th, 2015

9:00 — 9:30 Registration 9:30 — 9:45 Welcome of participants Willem Mulder (Utrecht)

Symposium 9:45 — 10:30 Keynote: DNA damage in preserved tissue specimens - sources, extent, counterme asures, Paweł Jałoszyński PhD (Wroclaw University)

Workshop

10:30 — 11:00 Opening of the specimen, vacuum filtration and dialysis, Natalia Winiarska MSc (Wroclaw Medical University) 11:00 — 11:30 Coffee break 11:30 — 12:00 DNA preparation from blood stain on the paper, Małgorzata Bonar MD (Wroclaw University, Institute of Anthropology), Miron Tokarski MSc (Wrocław Medical University), method developed at

Wrocław Medical University 12:00 — 12:30 Drilling of the bone and needle’s montage, Dominika Pluta, Tadeusz Dobosz Prof. (Wroclaw Medical University) 12:30 — 13:00 Montage of the system for DNA recovery from dental cave, Agnieszka Pilecka MD (Rex Company Wrocław) method developed at Wroclaw

Medical University

13:00 — 13:30 Covering the bone with silicone shell, Dominika Pluta, Tadeusz Dobosz Prof. (Wroclaw Medical University) 13:30 — 14:00 Finishing the DNA recovery from blood stain Małgorzata Bonar MD (Wroclaw University, Institute of Anthropology), Miron Tokarski MSc (Wrocław Medical University), method developed at

Wroclaw Medical University

Symposium

14:00 — 14:15 Results of nondestructive DNA preparation from human teeth, Agnieszka Pilecka MD (Rex Company Wrocław), method developed at

Wroclaw Medical University

14:15 — 14:30 Results of nondestructive DNA pepparation from blood stain Małgorzata Bonar MD (Wroclaw University, Institute of Anthropology), Miron Tokarski MSc (Wrocław Medical University), method developed at

Wroclaw Medical University

15:00 — 16:00 City tour — Museum of Pharmacy

Day 2 - Friday Septemeber 25th, 2015 Symposium 9:00 — 9:45 Keynote: aDNA in practice, Ryszard Słomski Prof. (Poznań University of Life Sciences) Workshop 9:45 — 10:15 Finishing the dental DNA preparation, Agnieszka Pilecka MD (Rex Company Wrocław) method developed at Wroclaw

Medical University

10:15 — 10:45 Coffee break 10:45 — 11:00 Finishing the DNA preparation from the dialysed conservation fluid, Natalia Winiarska MSc (Wroclaw Medical University) 11:00 — 11:45 Start of DNA preparation from a bone, Dominika Pluta, Tadeusz Dobosz Prof. (Wroclaw Medical University) 12:15 — 13:00 Lunch 13:00 — 16:00 City Tour 18:30 Banquet

Day 3 - Saturday Septemeber 26th, 2015 Workshop 9:00 — 9:20 Finishing of DNA preparation from a bone, Dominika Pluta, Tadeusz Dobosz Prof. (Wroclaw Medical University) 9:20 — 9:30 Results of the DNA preparation from wet museum specimens, Natalia Winiarska MSc (Wroclaw Medical University) 9:30 — 9:45 DNA purification on silicone columns, Arleta Lebioda MSc (Wroclaw Medical University) 9:45 — 10:15 DNA quantification, Małgorzata Małodobra-Mazur MD (Wroclaw Medical University) 10:15 — 10:45 Coffee break Symposium 10:45 — 11:15 Keynote: Landscape of aDNA testing, Tadeusz Dobosz Prof. (Wroclaw Medical University) 11:15 — 11:45 ABO and RhD blood groups determination from museum specimens, Małgorzata Małodobra-Mazur MD, (Wroclaw Medical University) 11:45 — 12:30 Documentary film: „Orgins of the DNA Sequencer” Ramunas Kondratas, (Vilnius University) 12:30 — 13:00 Closing remarks

ABSTRACTS

DNA damage in preserved tissue specimens—sources,

extent, countermeasures

Paweł Jałoszyński, PhD (Wrocław University)

Independently on the nature and source of museum or ancient tissue specimens, extracting and analysis of the genetic material encounters two major obstacles: contamination and DNA damage. While contami-nation issues are relatively similar to those affecting procedures involving modern and well-preserved samples, the DNA damage in historical and ancient material can accumulate to extents significantly inter-fering with amplification and sequencing, often causing false results. In living cells, exogenous (environmental) and endogenous (metabolic) factors continuously attack DNA and cause DNA damage, but this detrimental process is efficiently kept in check by specialized repair mechanisms. The repair, how-ever, ceases operating after the cell death, whereas in ancient and historical tissue specimens the DNA damage continues to occur and accumulates over time. DNA lesions, when damaged template is ampli-fied in the PCR reaction, may cause artificial mutations (when incorrect bases are incorporated opposite damage) or may inhibit or even entirely stop amplification, resulting in poor PCR yield. In ancient DNA, abasic sites generated by simple hydrolysis are predominating lesions, and they are both mutagenic and destabilizing DNA strands, being thus easily broken and representing the major cause of aDNA fragmen-tation. Common mutagenic and inhibiting lesions are oxidized nitrogen bases, accumulating in DNA ex-posed to atmospheric oxygen. One of the major problems and a significant source of sequencing errors is a hydrolytic deamination of cytosine, generating uracil, especially in single-stranded (ss) DNA (e.g., in overhanging ss ends of ds fragments). During amplification, templates containing deaminated cytosines produce artificial C to T substitutions, leading to erroneous results of detecting mutations in non-mutated target markers. A large number of DNA-DNA crosslinks was demonstrated to occur in tissues preserved in permafrost, leading to serious amplification problems, as they strongly inhibit DNA polymerases. A separate, but closely related problem, is an over 100-year old practice of preserving tissues in forma-lin. FFPE (formalin-fixed, paraffin-embedded) samples are valuable for a variety of molecular analyses, but formaldehyde inevitably generates DNA lesions. Formaldehyde-induced hydroxymethyl nitrogen base modifications, deaminated cytosines, abasic sites, strand breaks, DNA-protein and DNA-DNA crosslinks were demonstrated to account for up to 100% of sequencing artifacts in DNA extracted from FFPE speci-mens. Novel tissue fixatives, based on polyethylene glycol, may in future provide a non-damaging replace-ment for formalin. During extracting and amplifying DNA from historical and ancient samples, a number of precautions can be taken to avoid further DNA damage, and to reduce chances of sequencing artifacts. Handling sam-ples in oxygen-free atmosphere reduces additional oxidative damage; treatment with uracil-N-glycosylase eliminates uracil-containing tamplates; heat treatment cleaves formaline-induced DNA-DNA crosslinks; choosing short amplicons maximizes the number of templates in PCR, therefore reducing chances of se-quence artifacts; DNA polymerases with low damage by-pass efficiency will amplify only undamaged tem-plates; or special sequencing techniques can be used to detect low-frequency variants (i.e., misreplicated products) or use both DNA strands for cross-checking the sequencing results.

Nondestructive DNA isolation from old conservation fluids

Natalia Winiarska, MSc Wrocław Medical University

The purpose of the presentation is to present the nondestructive DNA isolation from old museum speci-

men’s preservative fluid. During the presentation will be shown two different methods of taking samples

and performing DNA isolation. The first one is the preservation fluid sample taking after fluid filtration

through filter paper. The second is the filter paper after filtration. The conservation fluid sample will be

purified by means of dialysis with the dialysis membrane and the paper filter – by use of QIAGEN Purifi-

cation Kit. The results will be obtained by PCR and capillary electrophoresis and will be presented by

marking AB0 blood groups, genotype and sex determination.

Nondestructive DNA isolation from blood stain on paper

Małgorzata Bonar MD (Wrocław University, Institute of Anthropology),

Miron Tokarski MSc (Wrocław Medical University)

Museum exhibits frequently offer valuable genetic information about owner or user. This information is

crucial to determine the person identity or to recognize possible illnesses that they was suffering from in

the past. Moreover obtaining genetic material from exhibits can lead to visual identification of the owner

based on specific markers. To that date the only way to obtain genetic material was inevitably connected

with the exhibit’s destruction. This situation in many cases prevented researches from conducting neces-

sary investigation. However, methods developed in Institute of Molecular Techniques, enable the non-

destructive procedure of DNA isolation from various museum samples.

This presentation will introduce method of nondestructive DNA isolation from blood stains that exist on

paper. Based on the own developed device that consists of low suction pump and heating element, to-

gether with special elution fluid, method in question enables high-performance DNA isolation from the

blood stain on the sheet of paper.

Nondestructive DNA isolation form bone

Dominika Pluta, Tadeusz Dobosz Prof.

(Wrocław Medical University)

Destructive method of DNA isolation, cause complete destruction of the bone. New method of

DNA isolation does not require grinding the bone. The method consist of drilling a chanel in the

bone - without entry into the medullary cavity. Then the needles are placed on both sides of the

channel. The entire bone should be covered with silicone. The 1-2ml of elution buffer was supplied

using linear syringe pump anly at +60C overnight. Obtained fluid was deproteinised by penthol-

chloroform extraction and purified using QIAquick PCR Purification Kit. The results obtained by the

destructive method were the same as non-destructive method.

Non-destructive method of DNA extraction from teeth by using QIAamp DNA Micro Kit Qiagen.

Agnieszka Pilecka MD (Rex Company Wrocław),

Tadeusz Dobosz Prof. (Wrocław Medical University)

DNA analysis can be used to widen our knowledge of extinct populations, world and human history. Den-

tal cavity often contains uncontaminated remains of ancient DNA. Archeological excavations are con-

venient source for various samples, but regretfully, in almost all extraction methods, a piece of bone or

tooth is powdered before extraction thus causing damage on archeological samples that are irreplacea-

ble and unique. We developed the method that allows us to obtain DNA from teeth preventing any sig-

nificant destruction of them. The system is based on rinsing dental root canal and dental cavity, that are

opened during decalcification process, by using perystaltic pump and a special buffer. The teeth remain

apparently intact and later can be stored in museum collections for ages.

Entire human teeth were used for a subject. Teeth were from fourteen to one thousand years old. Mod-

ern DNA was removed from the surface of the teeth by UV radiation. The first step of DNA extraction is

gentle decalcification with an 0,5M EDTA(Na2) buffer, pH 8.0, and the incubation of the tooth for five

days in a dark place. Next, the tooth is incubated in the commercial extraction buffer (Qiagen). I used

rubber hose with one end dipped in the buffer and the other end attached to a thin needle. The needle is

placed in the dental root canal, which is opened, in the preliminary decalcification. The hose is used in

conjunction with a peristaltic pump to make possible the slow flow of the extraction buffer through the

whole system. The dental root canal and dental cave are then rinsed for three days. After that, DNA is

separated from the buffer using commercial silica kit QIAamp DNA Micro Kit (Qiagen) [4]. The final elu-

tion volume is 30 microliters. Quantifiler® Human DNA Real Time Quantification Kit is used to quantify

the amount of amplifiable human DNA present in a sample. Isolated DNA is used in PCR to amplify five

STR loci. Primers and expected product size are given in Table 1. [6]

Table 1.

The amplifications are performed in a GeneAmp® PCR System 9700 (Applied Biosystems) with an 11 minute activa-

tion step at 95ºC, followed by 28 cycles at 95ºC for 60 seconds, 54ºC for 60 seconds, and 72ºC for 60 seconds. Up-

on completion of the 28 cycles the reaction is finished with 45 minutes of elongation at 60ºC. The amplified prod-

ucts are seperated by capillary electrophoresis using an ABI Prism® 310 Genetic Analyzer (Applied Biosystems).

Isolated DNA is used in mtDNA identification based on sequence differences in the hypervariable HV1 region. Pri-

mers and expected product size are shown in Table 2. [2].

Table 2.

STR name and chromo-somal location

Product size Allele Primers

AMEL

Xp22.1-22.3; Yp11.2

X- 56bp

Y- 62bp X,Y

5’-CCCTGGGCTCTGTAAAGAATAGTG-3’

5’-AGGACCACTTGAGAAAC-3’

D3S1358

3p21 90-126 bp 5-13

5’-TGACAGAGCAAGACCCTGTCTC-3’

5’-CATGAAATCAACAGAGGCTTGC-3’

TPOX

2p23-2 66-99 bp 5-13

5’-CAGGCACTTAGGGAACCCTCACTG-3’

5’-GTCCTTGTCAGCGTTTATTTGCCC-3’

D8S1179

8q24,1-24,2 70-106 bp 8-17

5’-GTATTTCATGTGTACATTCGT-3’

5’-ATTATTTTCACTGTGGGGAAT-3’

TH01

11p15-15,5 66-86 bp 5-10

5’-TTGCCTGTTCCTCCCTTATTTCCC-3’

5’-CACAAGGAACACAGACTCCATGGT-3’

Primer posi-tion

Product size Region Amplified Primers

L15971

H16258 287bp HV1a 15971-16258

5’-TTAACTCCACCATTAGCACC-3’

5’-TGGCTTTGGAGTTGCAGTTG-3’

L16140

H16414 274bp HV1b 16140-16414

5’-TACTTGACCACCTGTAGTAC-3’

5’-CACGGAGGATGGTGGTCAAG-3’

L00015

H00274 259bp HV2a 00015-00274

5’-CACCCTATTAACCACTCACG-3’

5’-TGTGTGGAAAGTGGCTGTGC-3’

L00145

H00389 244bp HV2b 00145-00389

5’-CTCATCCTATTATTTATCGC-3’

5’-CTGGTTAGGCTGGTGTTAGG-3’

The amplifications are performed in a GeneAmp® PCR System 9700 (Applied Biosystems) with a 15 mi-

nute initial denaturation step at 95ºC, followed by 32 cycles at 94ºC for 30 seconds, 57ºC for 90 seconds

and 72ºC for 90 seconds. Upon completion of the 32 cycles the reaction is finished by 10 minutes elonga-

tion at 72ºC.

After the detection of expected size PCR products in electrophoresis gel, PCR products are cleansed of

the primers and dNTPs using QIAquick PCR Purification Kit (Qiagen). Next PCR products are sequenced

using Big Dye Terminator v1.1 Cycle Sequencing chemistry, performed in a GeneAmp® PCR System 9700

(Applied Biosystems) the thermal cycling as follows: initial denaturation step at 96ºC for 60 seconds, 25

cycles consisting of denaturation at 96ºC for 10 seconds, annealing at 50ºC for 5 seconds and elongation

at 60ºC for 4 minutes. The products are purified using ethanol/sodium acetate precipitation before being

separated from the reaction products by electrophoresing on an ABI Prism® 3130 Genetic Analyzer

(Applied Biosystems) and analyzed by SeqScape v2.5 (Applied Biosystems) in comparison with the Cam-

bridge Reference Sequence.

REFERENCES

Hofreiter M., Serre D., Poinar HN., Kuch M., Pääbo S.: Ancient DNA. Nature Reviews: Genetics, Vol. 2,

2001; 353-359

Mitochondrial DNA Sequencing. Technical Booklet, The Perkin-Elmer Corporations.

Rohland N., Siedel H., Hofreiter M.: Nondestructive DNA extraction method for mitochondrial DNA ana-

lyses of museum specimens. BioTechniques, Vol. 36, 2004; 814-821

www.qiagen.com/literature/protocols/QIAampDNAMicro.aspx

www.products.appliedbiosystems.com

Żołędziewska M.: Badanie polimorfizmu mikrosatelitarnego w próbkach częściowo zdegradowanego

DNA z użyciem własnej metody reakcji multipleksowej PCR. Rozprawa doktorska, 2003.

aDNA in practice

Ryszard Słomski (1,2,3), Joanna Zeyland (1), Łukasz Wolko (1), Jan Bocianowski (1),

Marlena Szalata (1), Alexander M. Dzieduszycki (3), Mirosław S. Ryba (3),

Jan Śmiełowski 4), Daniel Lipiński (1)

1) Poznan University of Life Sciences, 2) Institute of Human Genetics, Polish Academy of Sciences 3) The Polish Foundation for Restoration of the Aurochs 4) Institute for Agricultural and Forest Environment, Polish Academy of Sciences

The beginning of molecular archaeology reaches the middle of the 1980s. Molecular archaeology includes analyses of ancient material on the molecular level, i.e. older than 75 years. The initial target was the isolation and characterization of the ancient DNA. In the course of time the next challenges were un-dertaken from which to the most important belong the research of DNA of extinct species and determin-ing their relationship with the today living species and also analyses of the relationship within the Homo genus in the aspect of the origin of the contemporary human. The first research concerning the aDNA from tissues of animals was conducted by Higuchi in 1984 and the research concerning a human was done by Pääbo in 1985 on the basis of the aDNA of Egyptian mummies. In the pioneering research, reaching a success was dependent on gaining relatively large quantities of DNA of good quality. The in-troduction to laboratories the polymerase chain reaction (PCR) which enabled to amplify DNA in vitro – popularized the aDNA research and enabled to conduct research also for the genetic material partially damaged as a result of the workings of environmental factors. One of the most important problems as-sociated with aDNA is DNA degradation following the death of a given organism as a result of action of endogenous nucleases. It is believed that, in conditions of physiological salt concentration, neutral pH and at the temperature of 15°C, after a passage of 100,000 years, hydrolytic processes destroy a DNA molecule completely. Very important is working with aDNA according to procedures given as the list of “authenticity criteria” and using archival material with possibility of receiving genetic material. The evi-dence showing authenticity of DNA coming from paleontological remains is its DNA sequence, very simi-lar but not identical to the DNA sequence of organisms with the same taxonomic affinity. At present, sci-entists express interest in the aDNA in the context of the influence of evolution on genetics of prehistoric populations.

Our contact with ancient DNA started very early with analysis of tooth of a man living 3,000 years ago, found in Warmian-Masurian Province in Poland during archaeological excavations. Now we are in-volved in analysis of an aurochs, a species similar to an African Buffalo, Water Buffalo, Muskox, Yak, Wi-sent or Bison. In case of an aurochs research is conducted on the basis of bones and teeth preserved in museum, private or recently found collections. The remains are early medieval materials and also are gained during digging peat or regulating rivers. To analyses come first of all fragments of cornual pro-cesses (beginnings of horns) and also teeth of an aurochs. For comparative research there are collected also samples of domestic and wild cattle. Material delivered to a laboratory is taken from the inside of bones, in order to limit possibilities of impurities by the contemporary DNA or impurities by conservants in which there was often bony glue applied. A surface of a bone can be additionally purified by fine sand-paper.

After gaining the aurochs’ aDNA a very important stage of work was to determine its quantity. The cloning of the aurochs’ DNA was conducted in order to duplicate the DNA isolated from bony mate-rial in the way that its quantities are fit to further works with identification of aurochs genes by a compar-ison of the results from our own research with the results placed in genetic data bases. To cloning there was used the DNA gained after the whole genome amplification (WGA). Sequencing of mtDNA of au-rochs allowed for obtaining of whole mitochondrial sequence (Bos primigenius mitochondrion, complete genome, GenBank: JQ437479.1)

DNA purification on silicone columns

Arleta Lebioda MSc (Wroclaw Medical University)

The QIAquick PCR Purification Kit (QIAGEN) was used for cleanup samples of DNA. Fragments

ranging from 100 bp to 10 kb were purified from rest of cells, previously used buffers, salts and proteinase

K. It’s a simple bind-wash-elute procedure with spin columns provided with silica-membrane and a mi-

crocentrifuge. Nucleic acids adsorb to the silica membrane in the high-salt conditions provided by the

buffer. Impurities are washed away and pure DNA is eluted with a small volume of low-salt buffer provi-

ded or water, ready to use in all subsequent applications including PCR and sequencing.

Now we try to replace this kit by similar kits produced by other manufacturer, for example EURx,

also.

Quantifier Human DNA Quantification Kit

Małgorzata Małodobra-Mazur, MD (Wrocław Medical University)

The Quantifier Human DNA Quantification Kit (Applied Biosystems; PN4343895) is design to quantify

the total amount of amplifiable human DNA in a sample. The results are mainly used to determine the

amount of sufficient human DNA that is capable to be used in short tandem repeats (STR) analysis as

well as to define how much sample is going to be used in STR applications. The kit is design for the ABI

Prism 7000 and Abi Prism 7900HT, both from Applied Biosystems.

The kit is composed of Human Primer Assay, Standard DNA and Reaction Mix. Human Primer Assay con-

sists of human DNA assay and internal control assay. The human DNA assay is in turn composed of a set

of primers and TaqMan probes labeled by FAM on 5’ end and non-fluorescent quencher at 3’ end. Addi-

tionally and the 3’ end TaqMan probes contain minor grove binder which increases melting temperature,

for that reason a shorter probes might be synthesized.

LANDSCAPE OF THE aDNA TESTING, FROM 1869 TO 2015.

Tadeusz Dobosz, Prof. (Wrocław Medical University)

Part I. Greatest discoveries in Molecular Biology

1869 F. Meischer prepared the „nucleine“ from cell nuclei

1889 R. Altmann renaned „nucleine“ into „nucleic acid“

1891 A. Kossel identified nucleic acid as a part of chromatine

1902 W. Sutton and T. Boveri discovered, that genes are located in chromosomes

1944 O.T. Avery, Mc. MacLeod and M. McCarty shoved, that nucleic scid transform bacteria

1950 E. Chargaff shoved, that nucleic acid from different species have different composition of

nucleotides

1952 A. Hershey and M. Chase discovered, that nucleic acids are the genetic material

1953 J. Watson, F. Crick and M. Wilkins described the helical DNA structure

1971 H.G. Khorana team described the PCR principle.

1971 P. Berg, W. Gilbert and F. Sanger developed the DNA sequencing

1981 S. Anderson published the mtDNA human genome

1983 K. Mullis developed the PCR method

2001 J.C. Venter and F. Collins published human haploid genome

2004 The FLX454, the first DNA next generation sequencer was introduced

2007 J.C. Venter published first human diploid genome

2010 S. Paabo team published the crude draft of neandertal genome

Part II. The history of aDNA testing

1981 first aDNA isolation

1984 first aDNA electrophoresis

1984 first aDNA sequencing

1988 first human aDNA sequencing

2010 first aDNA genome sequencing

Part III. Other nondestructive aDNA preparation approaches

2002 isolation from bored teeth nerve canal material

2004 isolation by gently bath the teeth in digestion fluid

2009 isolation of insect DNA by similar bath

2014 isolation from nerve cave over bored teeth holes

Part IV. Promising ideas for aDNA testing in future

2010 isolation aDNA by ethyl alcohol solutions

2011 testing aDNA without preliminary PCR

2015 use repair enzymes for improving the aDNA quality

Isolation of aDNA from museum specimens by first fully

nondestructive approach

Małgorzata Małodobra-Mazur, MD (Wrocław Medical University)

Museum specimens are with no doubts a valuable resource for the study of diverse histological process-es. Ancient DNA (aDNA) is enormously important in phylogenic and population genetic analysis. Unfor-tunately, most of the common DNA extraction methods are associated with tissue digestion and thus the precious museum specimens degradation. Applying non-destructive approaches for DNA extraction can help to save precious samples and simultaneously allow to obtain PCR capable DNA for further stud-ies.

ABO and RhD genotyping has been commonly used in phylogenic and population analysis. Furthermore, DNA analysis provides possibility for sex determination. Here we present a non-destructive method for DNA isolation from museum specimens and further applications that is ABO and RhD genotyping and sex determination at the molecular level.

The human tissues, the filter paper used to filter the conservation fluid and the dialyzed conservation flu-ids were examined for the ABO genotype and sex (Amelogenin) determination.

The results showed the same, replicable PCR profile - the same ABO blood group and sex determined by Amelogenin - in all three types of material from the same sample of tissue, filter paper and conservation fluid.

Wrocław Medical University

Institute of Molecular Techniques

M. Curie-Skłodowskiej 52, 50-369 Wrocław (Poland)

tel. 0048 71 784 15 88, fax: 0048 71 784 01 1

http://www.forensic.am.wroc.pl/index.php/zakad-technik-molekularnych