MTA BTK Régészeti Intézet - Archeogenetikai Laboratórium

Materials
Methods
Facilities

Methods



Sampling. Entering our clean pre-PCR laboratory, the bone samples get a unique identifier, they are registered and photographed. Whenever possible, two or three samples from anatomically distant locations are taken from each analysed skeletal remain. These samples are divided and treated independently.

Sandblasting A special isolated cabin serves for the primary mechanical cleaning of the bones. The entire cabin is regularly bleached and UV-C irradiated before and after the scrapings. The samples are also UV-C irradiated on all sides upon entering the lab. The surfaces of the bone samples are removed using a polisher machine. During the polishing, the bone powder by-product is persistently sucked off by a mobile vacuum cleaner. Tooth samples are cleaned with UV-C irradiation and sandblaster machine. The cleaned and subsequently UV-C irradiated samples are placed into sterile bags until further analyses.

The samples are ground into fine powder using a mixer mill (Retsch), measured in a sterile box and then stored in sterile tubes at 4°C until use. Each batch of samples has a negative control from the milling and later from the DNA extraction. The negative controls are processed parallel with the bone samples.

DNA Extraction The DNA extraction takes place in clean laboratory conditions, under a laminar flow box in a separate room. Several extraction methods are applied depending on the state of the sample and the purpose of the analysis. The DNA extracts, marked by specific ID-numbers, are stored in freezer.

A dedicated clean PCR-room is usedFor the preparation of PCR reactions and NGS DNA libraries, which is equipped with laminar airflow box.






Analysis of mitochondrial DNA (mtDNA)

PCR The mitochondria generate the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. The mitochondria have their own genome (mtDNA) with circular configuration. In contrast to nuclear chromosomal DNA, which is present in each somatic cell as a double set, mtDNA exists only as a single copy. It is inherited through the female line, because only the female mitochondria contributes to the newly developing individual's genetic makeup, and all members of a maternal lineage belong to the same mtDNA ancestry. Since several hundreds of mitochondria are found in a single cell, their DNA markers can be a useful source of information in maternal relationships, population genetics and history of populations.

Our primary focus was the Hypervariable Region I (HVR-I) of the mitochondrial genome, which is the most relevant and best studied part of the mtDNA in population genetic studies. We completed this analysis typing several polymorphisms in the coding region of the mtDNA, in order to reproduce our data and affirm certain haplogroup classifications. Since 2015, the PCR based methods have been gradually changed by whole mitochondrial genome sequencing, using target capture protocols and Illumina MiSeq sequencer.

Y-chromosomal DNA

Besides the small mitochondrial genome, the vast majority of the human genome is found in the cell nucleus. The nuclear genome is composed of 22 autosomes and two sex chromosomes. Since quantity (copy number) of the nuclear DNA is very small in the ancient samples, it is more challenging to study the chromosomes than the mtDNA. Out of the chromosomes, the Y-chromosome is inherited through the male line and has relatively few coding regions and high mutation rate. Most part of it (95%) does not recombine with the X-chromosome and therefore analysing its markers enables the follow-up of paternal lineages and male specific past population genetic events. The multiplex analyses of Y chromosomal SNPs (single nucleotide polymorphisms) and STRs (short tandem repeats) play significant roles in our studies. Since 2015 we have been using next generation sequencing techniques in these analyses as well.

Autosomal DNA

Post-PCR laboratory. In diploid human cells, the autosomes are composed of 22 chromosome pairs, each containing two variants of an allele or marker. The autosomes are commonly used in forensic science for identification and assignment of skeletal elements in disturbed burial sites, reconstruction of historic genealogies, and families. The best studied markers are the autosomal STRs, but also more and more highly polymorphic SNPs become frequently used. Besides ready to use commercial kits, we have been developing in house protocols for NGS (target capture) analyses of forensically and population genetically relevant markers. Furthermore, we also shotgun sequence the indexed DNA libraries.

Főoldal

Last update:
15.11.2018