sv.
Východisko. Problematika rozlišení genotypů matky a plodu v maternální plazmě těhotných žen je v současnosti řešena většinou pomocí real-time systémů. V těchto případech je rozpoznání genotypů možné použitím specifických sond, rozlišující jednotlivé genotypy. Nejčastější možnost se nabízí u gonozomálních sekvencí, kdy plod je mužského pohlaví. Tato práce popisuje možnosti detekce a kvantifikace fetální DNA pomocí analýzy STR lokusů. Metody a výsledky. K testování kvantifikačních možností kapilární elektroforézy (KE) byly použity arteficiální směsi genotypů v rozsahu 0,2 % - 100 %, které imitují genotyp matky a plodu. K detekci fetální DNA v maternální plazmě bylo použito 27 vzorků DNA těhotných žen v různém týdnu gravidity (t.g.). Genotyp plodu byl potvrzován genotypizací biologického otce. Detekce byla prováděna v STR lokusech z 21. chromozómu z oblasti zodpovědné za Downův syndrom (DS) metodikou inovované (I)QF PCR, která umožňuje zachytit a kvantifikovat i velmi vzácné mozaiky. Kvantifikace STR lokusů na KE byla posouzena na arteficiálních mozaikách a rozlišitelnost jednotlivých mozaik byla na úrovni několika procent. Fetální DNA byla detekována u 74 % testovaných vzorků. Závěry. Využití IQF PCR ke kvantifikaci a rozlišení maternálního a fetálního genotypu pomocí STR lokusů by mohlo mít význam v neinvazivní prenatální diagnostice jako další možný marker pro výpočet rizika DS.
Background. Problems of maternal and foetal genotype differentiation of maternal plasma in pregnant women are solved generally by real-time systems. In this case the specific probes are used to distinguish particular genotype. Mostly gonosomal sequences are utilised to recognise the male foetus. This work describes possibilities in free foetal DNA detection and quantification by STR. Methods and Results. Artificial genotype mixtures ranging from 0,2 % to 100 % to simulate maternal and paternal genotypes and 27 DNA samples from pregnant women in different stage of pregnancy were used for DNA quantification and detection. Foetal genotype was confirmed by biological father genotyping. The detection was performed in STR from 21st chromosome Down syndrome (DS) responsible region by innovated (I) QF PCR which allows to reveal and quantify even very rare DNA mosaics. The STR quantification was assessed in artificial mixtures of genotypes and discriminability of particular genotypes was on the level of few percent. Foetal DNA was detected in 74 % of tested samples. Conclusions. The IQF PCR application in quantification and differentiation between maternal and foetal genotypes by STR loci could have importance in non-invasive prenatal diagnostics as another possible marker for DS risk assessment.
- MeSH
- detekce genetických nosičů MeSH
- genom lidský MeSH
- lidé MeSH
- soudní lékařství metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
- Geografické názvy
- Česká republika MeSH
- Německo MeSH
In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.
- MeSH
- alely MeSH
- haplotypy * MeSH
- lidé MeSH
- lidský chromozom Y * MeSH
- mikrosatelitní repetice * MeSH
- soudní genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Sample containing 234 unrelated males and 197 unrelated females from Czech Republic was genotyped using an X-STR decaplex system in the following loci: DXS6789, DXS6809, DXS7132, DXS7133, DXS7423, DXS8378, DXS9898, DXS9902, GATA172D05, and GATA31E08. The linkage disequilibrium was observed between DXS6789 and DXS6809. The combined power of discrimination was 0.9999999998 (females) and 0.999998 (males). The mean exclusion chance was 0.999995 (trios) and 0.9998 (duos). This work presents the first population data for X-STR decaplex in Central Europe.
- MeSH
- DNA fingerprinting * MeSH
- etnicita genetika MeSH
- frekvence genu genetika MeSH
- genetická variace genetika MeSH
- genetické lokusy genetika MeSH
- genotyp * MeSH
- haplotypy genetika MeSH
- lidé MeSH
- lidské chromozomy X genetika MeSH
- mikrosatelitní repetice genetika MeSH
- populační genetika * MeSH
- vazebná nerovnováha genetika MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser-induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high-throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.
- MeSH
- algoritmy MeSH
- DNA rostlinná analýza genetika MeSH
- genetické markery genetika MeSH
- mikrosatelitní repetice genetika MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- reprodukovatelnost výsledků MeSH
- víno MeSH
- Vitis klasifikace genetika MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Cell-free fetal (cff) DNA analysis by short tandem repeats (STR) has the advantage of better recognizing the different genotypes. However, quantitative examination by quantitative fluorescent (QF) polymerase chain reaction (PCR) by STRs is limited to only a rough approximation. This project focuses on a more precise calculation of the relative cff DNA amount tested in the STRs' loci. METHODS: The cff DNA was analyzed in 363 samples from 258 pregnant women with physiological fetuses in different stages of pregnancy (from 4-37 gestational weeks) separately in three STRs [D21S1435, D21S1446 and PentaD (pD)] and also by gonosomal sequences amelogenin gene, X/Y-linked/testis specific protein, Y-linked (AMELX/Y/TSPY). Seventeen samples of cff DNA from fetuses with Down syndrome (DS) were compared. We optimized the refined quantitative fluorescent (RQF) PCR for STRs in a particular locus. RESULTS AND CONCLUSIONS: The cff DNA detection rate was 74% in at least one of the STRs. The efficiency decreased from shorter to longer PCR fragments. All three STR and gonosomal loci proved an increase in cff DNA during pregnancy. The stutter variability rate is greatest in short STR fragments and decreases as the STR fragments increase in length. Results showed that DS samples had a significantly higher amount of cff DNA.
BACKGROUND: Tetranucleotide Short Tandem Repeats (STRs) for human identification and common use in forensic cases have recently been used to address the population genetics of the North-Eastern Mediterranean area. However, to gain confidence in the inferences made using STRs, this kind of analysis should be challenged with changes in three main aspects of the data, i.e. the sizes of the samples, their distance across space and the genetic background from which they are drawn. AIM: To test the resilience of the gradients previously detected in the North-Eastern Mediterranean to the enlargement of the surveyed area and population set, using revised data. SUBJECTS AND METHODS: STR genotype profiles were obtained from a publicly available database (PopAffilietor databank) and a dataset was assembled including >7000 subjects from the Arabian Peninsula to Scandinavia, genotyped at eight loci. Spatial principal component analysis (sPCA) was applied and the frequency maps of the nine alleles which contributed most strongly to sPC1 were examined in detail. RESULTS: By far the greatest part of diversity was summarised by a single spatial principal component (sPC1), oriented along a SouthEast-to-NorthWest axis. The alleles with the top 5% squared loadings were TH01(9.3), D19S433(14), TH01(6), D19S433(15.2), FGA(20), FGA(24), D3S1358(14), FGA(21) and D2S1338(19). These results confirm a clinal pattern over the whole range for at least four loci (TH01, D19S433, FGA, D3S1358). CONCLUSIONS: Four of the eight STR loci (or even alleles) considered here can reproducibly capture continental arrangements of diversity. This would, in principle, allow for the exploitation of forensic data to clarify important aspects in the formation of local gene pools.
- MeSH
- frekvence genu * MeSH
- genetická variace * MeSH
- genotyp * MeSH
- mikrosatelitní repetice * MeSH
- populační genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- severní Afrika MeSH
- Střední východ MeSH
- Středomoří MeSH
Bones are a valuable source of DNA in forensic, anthropological, and archaeological investigations. There are a number of scenarios in which the only samples available for testing are highly degraded and/or skeletonized. Often it is necessary to perform more than one type of marker analysis on such samples in order to compile sufficient data for identification. Lineage markers, such as Y-STRs and mitochondrial DNA (mtDNA), represent important systems to complement autosomal DNA markers and anthropological metadata in making associations between unidentified remains and living relatives or for characterization of the remains for historical and archaeological studies. In this comparative study, Y-STR typing with both Yfiler™ and Yfiler™ Plus (Thermo Fisher Scientific, Waltham, MA, USA) was performed on a variety of human skeletal remains, including samples from the American Civil War (1861-1865), the late nineteenth century gold rush era in Deadwood, SD, USA (1874-1877), the Seven Years' War (1756-1763), a seventeenth-century archaeological site in Raspenava, Bohemia (Czech Republic), and World War II (1939-1945). The skeletal remains used for this study were recovered from a wide range of environmental conditions and were extracted using several common methods. Regardless of the DNA extraction method used and the age/condition of the remains, 22 out of 24 bone samples yielded a greater number of alleles using the Yfiler™ Plus kit compared to the Yfiler™ kit using the same quantity of input DNA. There was no discernable correlation with the degradation index values for these samples. Overall, the efficacy of the Yfiler™ Plus assay was demonstrated on degraded DNA from skeletal remains. Yfiler™ Plus increases the discriminatory power over the previous generation multiplex due to the larger set of Y-STR markers available for analysis and buffer modifications with the newer version kit. Increased haplotype resolution is provided to infer or refute putative genetic relationships.
- MeSH
- alely MeSH
- DNA fingerprinting přístrojové vybavení MeSH
- kosti a kostní tkáň chemie MeSH
- lidé MeSH
- lidský chromozom Y MeSH
- mikrosatelitní repetice * MeSH
- nekrotická degradace DNA MeSH
- oběti katastrofy MeSH
- polymerázová řetězová reakce MeSH
- tělesné pozůstatky * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
xii, 322 s. : tab., grafy ; 25 cm
