First experiences with the Spectrum Compact CE System

The newly released Spectrum Compact CE System by Promega is a capillary electrophoresis instrument developed for DNA-fragment separation and sequencing. In this study, its compatibility to 8 commercial short tandem repeat (STR) kits from 4 different manufacturers, reproducibility (sizing precision, accuracy and concordance) and robustness (sensitivity and mixture resolution) were tested and compared to the ABI PRISM® 310 Genetic Analyzer. The instrument was able to successfully analyse amplicons of all tested kits, proved to be as precise as claimed by manufacturer specifications and was shown to be more robust than the ABI PRISM® 310 Genetic Analyzer in some aspects. Analyses on the Spectrum Compact CE System were able to resolve peaks with length differences of 1-basepair in the short and long fragment range and mixtures of mixture ratios down to 1:30. We describe the advantages and limitations we have observed so far working with this instrument in our forensic genetics laboratory.

PEMERIKSAAN DNA SENTUHAN DARI BARANG BUKTI DENGAN PERBEDAAN JENIS BAHAN

Analysis of Touch DNA on forensic laboratory has been a favorite approach to identify a person. Every investigator demand the identity of whom the perpetrator that commit the crime, that leaved their DNA on the evidence. Many factors affect touch DNA, one of these is the substrate of the evidence. Common evidences that often examined in forensic lab are firearms, knife, swords, clothes, and switch bomb. To collect the cell on the evidence we use tapelift method using the duct tape. PrepFilerTM BTA Extraction Kit used to extract the DNA from the duct tape, followed by Quantifiler® Duo. For profiling the DNA we use GlobalFilerTM and fragment analyzed on ABI 3500 Genetic Analyzer followed by GeneMapper ID.X. V.1.4. Based on our analysis, DNA from fabric substrate has the higher percentage of success DNA profiling. The success DNA profiling rate of fabric and plastic substrate is 100%, and 0% for wood substrate. According to recent researches, smooth substrate, like plastic and glass, has higher percentage to get full profile than rough substrate, like woods. But on the fabric, they found has much higher percentage than smooth substrate. This can be due to the absorption ability of the fabric to obtain more cells

PSVIII-2 Genetic diversity analysis of hybrid Karachaev goats (Capra hircus) population using microsatellite marker data

The Karachaev goat is an indigenous breed that possesses unique features including significantly less fat deposition in the body compared to sheep and cattle, ability to graze at an altitude of up to 1200 meters and to produce fertile hybrids with wild relatives. To understand the genetic diversity and population structure of hybrids between domestic Karachaev goats (Capra hircus) and The West Caucasian tur (Capra caucasica) 143 individuals were analyzed using 10 microsatellites panel. Sample were analyzed according to the manufacturer’s recommendations on an automatic sequencer, ABI 3130XL genetic analyzer (Applied Biosystems). Genetic diversity was calculated using GenAlEx 6.503 software. Genotyping of ten microsatellite loci in hybrid forms of Karachaev goats and turs detected 106 alleles in total. Na values ranged from five (INR063) to seventeen (SRCRSO008), averaging 10.6 alleles per locus across the 10 loci. All loci were polymorphic. The average number of alleles was 10.6 alleles per locus. This is higher than the similar indicator obtained by Kharzinova et al. (2019) in populations of Soviet wool, Tajik wool, Orenburg downy, Alpine and Zaanen dairy breeds of goats, studied using the panel of same 10 STR-markers (9.3 loci). Other key indexes of genetic diversity could be found in table 1. The values of the coefficient FIS suggest the absence of related mating in the herd. Information on genotypic variability of Karachaev goats hybrid forms obtained here will contribute into the breeding programs improvement and to preservation of existing native breeds.

Development of 3 Multi-PCR Based Microsatellite Instability Analysis (MSA) for Bladder Cancer Detection

Several studies have shown that microsatellite changes can be profiled in urine for the detection of bladder cancer. Use of microsatellite analysis (MSA) for bladder cancer detection requires a comprehensive analysis of up to 15 to 20 markers, which were based on amplification and interpretations of many individual MSA markers and can be technically challenging. Here, in a way to develop fast, more efficient, standardized and less costly MSA for detection of bladder cancer, we have developed 3 multiplex PCR based MSA assay, all of which were analyzed by genetic analyzer. First, we have selected 16 MSA markers based on 9 selected publications. Based on samples from Johns Hopkins University (JHU Sample, first set sample), we have attempted to develop MSA assay based on doublet, 2 tube based multiplex PCR combined with singlet, 1 tube based single plex PCR. While this assay was initially successful, we have encountered a reproducibility issue and we then developed MSA based on triplet, 3 tube based multiplex PCR (Triplet MSA assay). From second set samples (6 cancer patients’ and 14 normal individuals’ sample), our Triplet Assay with 15 MSA markers correctly predicted all of 6/6 cancer samples to be cancerous sample and 14/14 samples from normal individuals as normal samples. This result suggests that our Triplet MSA Assay combined with genetic analyzer is a potentially time and cost-effective genetic assay for bladder cancer detection and can be used potentially as a dependable assay in patient care.

PSXIV-25 Search for the association of SNP in GRM8 gene with sperm quality in stallions

The formation and functioning of the animal reproductive system occurs as a result of the coordinated interaction of a wide range of genes. To search for causal mutations, work was carried out to search for polymorphic variants in the marker regions detected using GWAS. The four single-nucleotide substitutions in the exon of the GRM8 gene identified during the studies and the association of these SNPs with sperm quality was carried out. Semen from 22 stallions was collected. Sperm volume, concentration and progressive motility were assessed. Sequencing of the sections of the candidate GRM8 gene was carried out using an Applied Biosystems 3500 genetic analyzer. For the rs1138419111 genotype, no significant differences were found in the studied parameters. According to the identified single nucleotide substitution rs1147388106, the highest ejaculate volume was in stallions with the GG genotype (55.9±26.5 ml) compared to stallions with the GA genotypes (32.5±13.9 ml) and AA (18.0±33,6) (p < 0.05). When analyzing data on SNP rs395286150, stallions with a heterozygous CT genotype had the best sperm quality. Thus, the cell concentration was 317.0±66.5 million/ml in stallions with the CT genotype, 209.6±58.2 and 189.5±74.9 % with the CC and TT genotypes, respectively (P < 0.05). The progressive sperm motility of stallions with the CT genotype was 65.5±20.5% versus 48.7±22.0% in stallions with the TT genotype and 48.4±18.6% with CC. Analysis of data on SNP rs394524550 revealed a significant effect of the genotype on progressive motility. Stallions with the AG genotype had a progressive motility of 64.6±16.3%, and those with GG and AA 32.7±15.7 and 49.6±18.1%, respectively (P < 0.05). Thus, as a result four single nucleotide polymorphisms were identified in the exon of the GRM gene. Three of them were significantly associated with such indicators of sperm quality as ejaculate volume, concentration and progressive motility. Project No. 0445-2021-0011.

Genetic identification of the causative agent of Brucellosis

The development of animal husbandry suffers various kinds of losses due to the spread of infectious diseases among animals, in particular Brucellosis. A challenge faced by Brucella researchers has been the accurate identification of new isolates within the genus while preserving sufficient, and not excessive, biosafety and biosecurity requirements. The availability of discriminatory molecular typing tools to inform and assist conventional epidemiological approaches would be invaluable in controlling these infections, but efforts have been hampered by the genetic homogeneity of the genus. In this work, for better identification of infection, for control and monitor the source of outbreaks in prosperous areas was carried out identification of Brucella spp. strains which circulating in the Kostanay region. For this was used using multilocus analysis of a variable number of tandem repeats sequenced by 16 s – PNK on a genetic analyzer (sequencer). According to the results of a study of cattle, cultures of microorganisms were infected: No. 4, 5, 7, 8. Comparison of the obtained results with similar results of domestic and foreign works by A. Shevtsov, G. Borrello, P. Le Fleche, G. Garofolo suggest that the genotyping of local strains has an importance in the molecular epizootology of the Republic of Kazakhstan.