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Optimization of digital polymerase chain reaction for application in non-invasive prenatal diagnostics
Šenkyřík, Pavel ; Korabečná, Marie (advisor) ; Vodička, Radek (referee)
Digital PCR (Polymerase Chain Reaction) is a method that enables absolute quantification of DNA sequences and therefore finds application in many diagnostic disciplines. We focused on the diagnosis of trisomy 21, which manifests itself as Down syndrome. Information about the fetal genome can be obtained from free fetal DNA that is released into the mother's bloodstream from the placenta during pregnancy. In the first trimester, fetal DNA constitutes approximately 5-10% of free DNA and is therefore a suitable target for non-invasive prenatal testing (NIPT). However, we need a technique that is able to distinguish this relatively small fraction of free fetal DNA and differentiate it from free maternal DNA. Precisely digital PCR provides various ways to achieve this goal thanks to its wide multiplexing possibilities. We focused on optimizing the multiplex reaction, which in one fluorescence channel distinguishes the number of copies of the reference chromosome 18 and the observed chromosome 21. The resulting determination of this ratio provides us with information regarding the balance between the chromosomes and can thus confirm or refute the presence of a trisomic fetal fraction. We tested the usability of the proposed method for clinical application by examining DNA samples isolated from 25 plasma...
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Detection of CNV polymorphisms in regions of forensically significant STR loci in the Czech population
Salihagić, Lejla ; Korabečná, Marie (advisor) ; Šimková, Halina (referee)
DNA analysis aimed at individual identification of persons is based on the examination of a panel of STRs (Short Tandem Repeats). These polymorphic loci were selected on the basis of broadly conceived international testing and the frequencies of individual alleles across different ethnicities are known. Currently, attention is also paid to polymorphisms involving larger regions of DNA - the so- called Copy Number Variants (CNV). According to the literature (Repnikova et al., 2013), it turns out that these CNVs can also affect areas of forensically significant STR loci, resulting in, for example, the deletion of one of the alleles. The examined person, then in such an affected locus is not a homozygote, but a hemizygote. Otherwise, when duplication event of one of the alleles occurs, a tri-allelic STR arises. Such situations can then distort the result of individual identification, or even the correct evaluation of kinship relations. In the scope of master's thesis, a methodology based on digital droplet PCR (ddPCR) will be established, which will allow verification in cases where routinely obtained DNA profiling results in the suspicion of the presence of a third allele in one of the STR loci. Samples for testing will be obtained in cooperation with the Institute of Criminology in Prague on the...
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Association study with the ADA gene and detailed characterization of two chromosome aberrations in autism
Vážna, Alžběta ; Sedláček, Zdeněk (advisor) ; Ferák, Vladimír (referee) ; Korabečná, Marie (referee)
Autism affects up to 1/150 children and represents therefore a serious social problem. It is a complex disorder with a clearly documented genetic component, but so far unexplained aetiology, which is currently a subject of intensive research. In the field of genetics various gene and chromosome defects are examined, as well as other mechanisms, including epigenetics, which could play a role in pathogenesis. In our work we tried to replicate the finding of association between the ADA*2 risk allele and autism in a sample of 385 Czech children. Our sample was larger than those originally published. We also focused on individual endophenotypes (types of autism, degree of mental retardation and co-morbidity). Our results did not confirm the association of autism with the ADA*2 allele in the complete sample or in any of the subsets. Chromosomal changes represent another finding in autistic patients. We performed the analysis of a ring chromosome 17 and a chromosome X deletion in two patients. Our studies represent an up to now unimaginable link between classical cytogenetics and molecular genetics at the DNA sequence level. As the first in the world, we described the structure of a human ring chromosome. Characterization of the defects allowed us to speculate on the impact of the genes involved in the phenotype...
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MicroRNAs in AML pathogenesis
Koutová, Linda ; Korabečná, Marie (advisor) ; Zemanová, Zuzana (referee) ; Kotyza, Jaromír (referee)
Acute myeloid leukemia (AML) is a very heterogeneous disease associated with cytogenetic aberrations and genetic mutations. Many of these changes have been revealed and their detection became usual part of the diagnostic process today. However, changes of expression profiles of small, noncoding RNAs, so called microRNAs (miRNAs), are less known and not used for diagnostics yet. These RNAs, 19-24 nucleotides long, take part in the regulation of expression of different genes through complementary base pairing to the 3'non- translated region (3'UTR) of the target messenger RNA (mRNA). They can influence key processes of the cell, like differentiation, proliferation or apoptosis. The changes in expression of different miRNAs are known from different types of cancers. In solid tumors, they are usually detected from bioptic samples; but also plasma samples are now in the center of attention as so called liquid biopsies providing the information about molecular genetic events in the organism. Many studies have revealed deregulated miRNAs in the bone marrow, full blood or isolated progenitor cells (CD34+) of AML patients, only four of them have analyzed plasma samples. We focused on the plasma samples and we targeted on such miRNAs, which levels differ at AML diagnosis and after the chemotherapy. Out of...
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Role of polymorphic DNA markers in personal identification and determination of selected phenotypic traits
Zidkova, Anastassiya ; Korabečná, Marie (advisor) ; Drábek, Jiří (referee) ; Hradil, Roman (referee)
Nowadays intensive research is conducted for application of genetic polymorphisms for degraded samples analysis, identification and kinship determination. Another area of research in forensic genetics is biogeographical and phenotypic traits (eye, hair and skin color) determination. First part of presented work dealt with population study on the Czech popu- lation using Investigator DIPplex (QIAGEN, Germany) marker set containing 30 autosomal insertion-deletion polymorphisms. Power of Discrimination (PD), which is the probability of random selection of two persons with different genotypes, was 99.9999999999% for the whole marker set. This part of study concluded that ana- lyzed marker set is suitable as an additional marker panel for identification and kinship determination in the Czech Republic. Second part of the presented study was devoted to population research of Cen- tral Croatia using Mentype Argus X-8 kit (QIAGEN, Germany) containing 8 short tandem repeat polymorhisms located on X choromosomes (X-STR) divided into 4 linkage groups. PD for the whole kit reached 99.9999% and 99.99999999% for males and females, respectively. This kit could be used in Central Croatian population for kinship analysis and for identification as an additional marker panel. The next part of the presented study was the...
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Forensic SNP identification of trichological material by High Resolution Melting analysis
Strobachová, Joanne ; Stenzl, Vlastimil (advisor) ; Korabečná, Marie (referee)
The analysis of mitochondrial DNA (mtDNA) from shed hairs has gained high importance in forensic casework since telogen hairs are one of the common type of evidence left at the crime scene. This type of evidence very often lack of nuclear DNA (nDNA), so the analysis of mtDNA is the only possibility. The analysis of trichological material lacking nDNA is not commonly done in the forensic practice because of the high price of the regularly used mtDNA analysis method, as well as the time demandingness. For these reasons we decided to analyze the mtDNA and identify the trichological material by analysis of chosen single based polymorphism's (SNP) followed by High Resolution Melting (HRM). In order to verify the robustness of the HRM analysis method on several types of biological material that belongs to one person, we tested trichological material in anagen and telogen. From total number of 5 selected SNP's, this paper was able to optimize analysis of 3 SNP's. Due to the analysis of SNP C14766T (applicable for the trichological material in anagen and telogen), SNPs C7028T and G3010A (applicable only for the trichological material in anagen). The mtDNA sequences of different biological materials did not show intra-individual differences. Protocol that could be used for the forensic practice with the use...
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Utilisation of molecular cytogenetic techniques in productive genetics
Paulasová, Petra ; Macek, Milan (advisor) ; Korabečná, Marie (referee) ; Vodička, Radek (referee)
Title.: Utilisation of molecular cytogenetic techniques in reproductive genetics Chromosomal abnormalities constitute one of the most important causes of birth defects, fertilization failure and/or human infertility. Approximately, 40-50% of human conceptuses are chromosomally abnormal, 6% of the abortions during the first trimester of gestation are directly linked to chromosomal abnormality, while at term 0.6% of livebirths present with such features. Most of these abnormalities originate from the gametogenesis and arise through disturbed meiotic processes. Each gamete is a final and original product of the meiosis carrying a unique chromosomal set. Therefore, cytogenetic examination of individual gametes represents an important scientific challenge for our undrstandidng of the formation, incidence and etiology of aforementioned chromosomal abnormalities. Nonetheless, it is very technically demanding to perform efficient chromosomal investigation on gametes, hence single cells. My Ph.D. thesis is focused on the development of new techniques for the detection of chromosomal abnormalities in gametes and embryos. We developed a PNA-based technique as an alternative to conventional FISH and PRINS-based methods for fast, efficient and robust in situ detection of chromosomal abnormalities in human...
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The function of telomeres and cell-free DNA in the healthy volunteers and patients with chosen pathological condition
Zinková, Alžběta ; Korabečná, Marie (advisor) ; Vodička, Radek (referee) ; Drábek, Jiří (referee)
More than 70 years have passed since the discovery of cell-free DNA (cfDNA), but the greatest interest in this topic and knowledge has undoubtedly occurred in the last thirty years. It is used mainly in oncology and prenatal diagnostics. While it is routinely used diagnostically in these fields, little is known about its physiological functions in the organism. Our research therefore focuses on understanding this role and in experiments works mainly with samples obtained from healthy individuals. The first study focused on the differences between plasma and serum in healthy individuals. We asked the question whether they differ in cfDNA concentration and telomeric sequences abundance. We found that the serum contains significantly more cfDNA than plasma, on the other hand, plasma is relatively richer in telomeric sequences. In stimulation experiments with THP1 cells, samples cultured with DNase-treated serum (without cfDNA) showed a higher expression of mRNA TNF-α (Tumor necrosis factor α) than samples untreated. The same trend was observed when plasma samples were stimulated. A study involving plasma samples from ten patients with celiac disease and ten healthy controls showed significant differences in mRNA TNF-α expression between experiments in which THP1 cells were stimulated by DNase-treated...
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Postpartum expression of cardiovascular disease-associated microRNAs - comparison of expression levels between plasma, plasma exosomes and whole peripheral venous blood
Ševčíková, Adéla ; Hromadníková, Ilona (advisor) ; Korabečná, Marie (referee)
MicroRNA (miRNA) are short non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Many miRNAs are involved in the pathogenesis of cardiovascular diseases, which is associated with altered gene expression. This work compares miRNA gene expression profiles among various biological sources - whole peripheral venous blood (whole PB), plasma and plasma exosomes. For all tested groups combined, the expression levels of miRNA were maximal in whole PB and lowered in plasma and plasma exosomes, and the expression levels of miRNA were higher in plasma than in plasma exosomes, except miR-126-3p, which had a higher level detected in plasma exosomes compared to plasma. This work also compares expression levels of cardiovascular miRNA between women with anamnesis of gestational diabetes mellitus (GDM) and physiological gravidity 3-11 years postpartum in whole PB, plasma and plasma exosomes. In whole PB, 12 of 29 tested miRNAs were up-regulated in women with prior exposure to GDM. MiR-181a-5p was up-regulated in plasma exosomes and miR-499a-5p in plasma in women with prior exposure to GDM. The changes in whole peripheral venous blood seem to reflect the complex systemic response to the changes that occurred during the onset of GDM. Women with aberrant epigenetic profiles may...
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