Specific PCR Products Research Articles

Novel KRAS gene mutations in Saudi colorectal cancer patients

We report 7 novel KRAS gene mutations discovered while retrospectively studying the prevalence and pattern of KRAS mutations in cancerous tissue obtained from 56 Saudi colorectal cancer patients from the Eastern Province. Methods Genomic DNA was extracted from paraffin-embedded, formalin-fixed cancerous and noncancerous colorectal tissues. A nested PCR approach was implemented. Successful and specific PCR products were then bi-directionally sequenced. The functional impact of the novel mutations on the K-ras protein was assessed using bioinformatics tools and molecular modeling. Results KRAS gene mutations were detected in the cancer tissue of 24 out of 56 cases studied. Of these, 11 were in exon 4 (19.64%). The 11 cases with exon 4 aberrations harbored 8 different mutations. All of these except two altered the K-ras protein amino acid sequence and all except one were novel as revealed by COSMIC database. The detected novel mutations were found to be somatic. The molecular modeling data fit with the prediction from Polyphen-2 (polymorphism phenotyping v2) and SIFT (sorting intolerant from tolerant tools), as well as conservation data. One mutation is predicted to be benign and modeling also showed little predicted effect on protein structure. The remaining mutations are predicted to cause substantial changes in the protein structure in line with the predicted damaging effect by polyphen-2 software. Of these, the Q150X nonsense mutation is the second truncating mutation to be reported in colorectal cancer in the literature.1 Conclusions Our discovery of novel exon 4 KRAS mutations that are, so far, unique to Saudi patients from the Eastern Province may be attributed to environmental factors and/or racial/ethnic variations due to genetic differences. Alternatively, it may be related to the paucity of clinical studies on mutations other than those in codons 12, 13, 61 and 146. Further KRAS testing on a large number of patients, particularly beyond the most common hotspot alleles in exons 2 and 3, is needed to assess the prevalence and explore the exact prognostic and predictive significance of the discovered novel mutations as well as their possible role in colorectal carcinogenesis.

Peptidoglycan Hydrolases as Species-Specific Markers to Differentiate Lactobacillus helveticus from Lactobacillus gallinarum and Other Closely Related Homofermentative Lactobacilli

We propose a new method that allows accurate discrimination of Lactobacillus helveticus from other closely related homofermentative lactobacilli, especially Lactobacillus gallinarum. This method is based on the amplification by PCR of two peptidoglycan hydrolytic genes, Lhv_0190 and Lhv_0191. These genes are ubiquitous and show high homology at the intra-species level. The PCR method gave two specific PCR products, of 542 and 747 bp, for 25 L. helveticus strains coming from various sources. For L. gallinarum, two amplicons were obtained, the specific 542 bp amplicon and another one with a size greater than 1,500 bp. No specific PCR products were obtained for 12 other closely related species of lactobacilli, including the L. acidophilus complex, L. delbrueckii, and L. ultunensis. The developed PCR method provided rapid, precise, and easy identification of L. helveticus. Moreover, it enabled differentiation between the two closely phylogenetically related species L. helveticus and L. gallinarum.

Assessment of PCR-based tools for the specific identification of some temperate Meloidogyne species including M. chitwoodi, M. fallax and M. minor

Several conventional PCR tests have been developed for the identification of the European quarantine root-knot nematodes Meloidogyne chitwoodi and M. fallax but data are lacking for the evaluation of their performance in terms of sensitivity, repeatability, reproducibility and specificity against a large range of populations. This study evaluated the performance criteria of three conventional PCR tests recommended by the consensus diagnostic protocol for Meloidogyne chitwoodi and Meloidogyne fallax published by the European and Mediterranean Plant Protection Organization (EPPO): a species-specific PCR (IGS target), a SCAR PCR, and a rDNA ITS PCR-RFLP. Evaluation was carried out with DNA extracts from juveniles, males and females according to EPPO recommendations for test validation. A minimum of 34 populations of target and non target nematode species were tested to check the specificity of these three PCR assays. The three PCR tests were ranked according to their specificity (with regard to cross reaction with other nematodes species or genus) and their sensitivity (detection of a single juvenile or mixed with other species). The species-specific PCR proved to be more sensitive but less specific than the SCAR PCR. The PCR-RFLP enables the identification of several Meloidogyne species but profile analysis can be difficult when several species are present in the mixture. Specific PCR products and RFLP profiles were also observed for M. arenaria and M. enterolobii, and described for M. minor and M. artiellia.

Molecular and parasitological study of cutaneous leishmaniasis in Bushehr province, southwest of the Islamic Republic of Iran: a cross-sectional study during 2009-2012.

Cutaneous leishmaniasis (CL) is one of the most important parasitic disease in Iran. CL is distributed among more than half of 31 provinces of Iran. Studies on epidemiological aspects of the disease and Leishmania species identification among infected humans are necessary for providing a comprehensive prevention and control program thus; this descriptive cross-sectional study was conducted on all CL suspected patients who referred to Health Centers of Bushehr province from 2009 to 2012. Physical examinations were carried out in suspected individuals and CL cases were confirmed by microscopical examinations. Prepared slides from suspicious cases of CL were fixed with absolute methanol and stained by Giemsa 10%. All the Giemsa-stained slides examined under a light microscope with high magnification (1,000×) and classified them based on grading of Leishmania parasites. DNA from each slide was extracted, separately. The ribosomal internal transcribed spacer 1 was amplified with specific primers and PCR products were digested by restrict enzymes (HaeIII), run them in 3% gel agarose for electrophoresis and visualized on a UV transilluminator after staining with ethidium bromide. SPSS version 21 was used for data analyses. A total of 726 suspected CL cases were referred to Health Centers of Bushehr province from 2009 to 2012 and samples were only prepared from 188 of the patients whereas 43 (5.9%) of them were microscopy positive. The most frequent of CL was observed in November (14%) and December (12%). The most distribution of CL lesions were observed on hands (32%), feet (26%), and face (21%), respectively. The highest frequency of CL was observed in 1-9years old (30%). Altogether, 50% of the patients showed one skin lesion and 2-10 skin lesions were occurred in the remained CL patients. Totally, 27 out of 43 (63%) of the Giemsa stained slides were positive by PCR-RFLP assay because all the PCR-RFLP negative slides were prepared 3-4years ago and kept without cover slip, and also observed scarce amastigotes during microscopy observations. Leishmania species were identified in 21 desirable slides which 14 of them were L. major and 7 of the remained isolates were identified L. tropica using PCR-RFLP.

Solid-phase plate-reader quantification of specific PCR products by measurement of band-specific ethidium bromide fluorescence

Real-time PCR is widely employed to quantify PCR products across a range of applications. However, accurate real-time PCR is not always technically feasible, and alternative methods for PCR product quantification can be expensive and time consuming to validate. We have developed an inexpensive, rapid, and immediately accessible protocol to quantify PCR products, by measuring ethidium bromide fluorescence of PCR products excised from agarose gels. This protocol has relevance to a broad range of methods in molecular biology where quantification of PCR products is necessary.

Identification and application of a unique genetic locus in diagnosis of Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium oxysporum f. sp. cubense, particularly the tropical race 4 (TR4), is the most destructive fungal pathogen of banana and can cause significant economic losses. However, the molecular mechanisms underlying the pathogenicity and virulence of the fungus are poorly understood. In this study, we characterized the mutant W2987, which was derived through T-DNA insertional mutagenesis of a wild-type TR4 isolate. W2987 showed slower growth, produced fewer conidia and was less virulent than the wild-type strain. Thermal asymmetric interlaced-PCR (TAIL-PCR) analysis showed that the T-DNA was inserted in the 3′ untranslated region (UTR) of an open reading frame (ORF) in W2987. BLAST search results indicated that the ORF encodes a hypothetical protein with unknown function. Phylogenetic analysis of the gene sequences from various Fusarium isolates showed that the F. oxysporum f. sp. cubense TR4 isolates were distinct from other F. oxysporum isolates. One pair of primers based on the 3′ UTR sequence of the putative virulence gene amplified a specific PCR product from the F. oxysporum f. sp. cubense TR4 isolates but not from other races and Fusarium species. This result indicates that this genetic locus possibly associated with virulence is unique and reliable for diagnosis and detection of F. oxysporum f. sp. cubense TR4.

Investigation of MGMT and DAPK1 methylation patterns in diffuse large B-cell lymphoma using allelic MSP-pyrosequencing.

The tumor suppressor genes MGMT and DAPK1 become methylated in several cancers including diffuse large B-cell lymphoma (DLBCL). However, allelic methylation patterns have not been investigated in DLBCL. We developed a fast and cost-efficient method for the analysis of allelic methylation based on pyrosequencing of methylation specific PCR (MSP) products including a SNP. Allelic methylation patterns were reliably analyzed in standards of known allelic methylation status even when diluted in unmethylated DNA to below 1% methylation. When studying 148 DLBCL patients MGMT and DAPK1 methylation was observed in 19% and 89%, respectively, and among methylated and heterozygous patients 29% and 55%, respectively, were biallelically methylated. An association between the T-allele of the rs16906252 SNP and MGMT methylation was observed (p-value = 0.04), and DAPK1 methylation of the A-allele was associated with shorter overall survival (p-value = 0.006). In future cancer research allelic MSP-pyrosequencing may be used to study a wide range of other loci.

PCR-based segregation of one hybrid variety of Labeo rohita and Catla catla from their wild-types

High consumer preference together with its polyculture potential has undoubtedly driven Rohu (Labeo rohita) and Catla (Catla catla) to top the list of the most preferred fishes among the Indian major carps. Commonly found in these fishes are hybrids that can be natural or man-made. Increasing emphasis on biodiversity issues has necessitated proper stock management of these through molecular genetics techniques. Also with few morphological differences that can be used to differentiate wild types and hybrids properly, the problem demands a straightforward molecular approach. Here, we report a simple PCR-based technique that can differentiate the hybrid variety from wild types easily using three different microsatellite markers. Three sets of primers were used to amplify three different microsatellite markers from the genomic DNA isolated from pectoral fins. When the PCR products using all three primer sets were analyzed, ‘hybrid–Rohu’ could be distinguished from wild types. Whereas the hybrid–Rohu DNA yielded specific PCR products with all three primer pairs, only two PCR products were obtained either from wild-type Catla DNA (by primer sets 1 and 2) or from wild-type Rohu DNA (by primer sets 1 and 3). This study clearly demonstrates that a simple PCR-based technique will help the fish breeders and hatcheries to identify and differentiate suspected hybrid–Rohu carp from the wild types within a few hours.

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

Detection of Salmonella in Chicken Meat by Insulated Isothermal PCR

Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfH gene encoding a heat shock protein for the iiPCR detection of Salmonella in chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5' and 3' ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonella tested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonella strains tested, including strains of Enterobacteriaceae closely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonella at as low as 10⁰ CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps.

Abstract B28: Investigation of MGMT methylation patterns in diffuse large B-cell lymphoma using a novel allelic methylation-specific PCR pyrosequencing assay

Abstract MGMT is a tumor suppressor gene, which becomes methylated in many different cancers including diffuse large B-cell lymphoma (DLBCL). Methylation of MGMT has been associated with the T-allele of the rs16906252 SNP found in the MGMT promoter. However, allelic MGMT methylation patterns and a possible association with the T-allele of this SNP have not been investigated in DLBCL. Today, standard treatment for DLBCL patients is rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), and methylation of MGMT has been shown to predict chemosensitivity to the alkylating agent cyclophosphamide in the era before rituximab. We developed a novel method for the analysis of allelic MGMT methylation patterns based on pyrosequencing of antisense methylation specific PCR (MSP) products including the rs16906252 SNP. Samples from 148 DLBCL patients, of which 75 received R-CHOP, were studied using this assay. Standard bisulfite pyrosequencing and bisulfite sequencing of single clones were used to confirm data obtained by allelic MSP pyrosequencing. The allelic MSP pyrosequencing assay could reliably analyze allelic methylation patterns in standards of known allelic methylation status even when diluted in unmethylated DNA down to 1.25% methylation. MGMT methylation was observed for 28 patients of which seven were heterozygous. Thus, an association between the T-allele and MGMT methylation was observed (p-value =0.04). Among the seven methylated and heterozygous patients, two were methylated at both alleles, four were methylated only at the T-allele, and one only at the C-allele. Finally, we found no statistical significant associations between MGMT methylation or SNP genotypes with clinical parameters among the R-CHOP treated patients. In conclusion, allelic MSP pyrosequencing is a fast and cost-efficient method for evaluation of allelic methylation patterns, which may have wide implications for locus specific methylation analysis of tumor suppressor genes in future cancer research. Citation Format: Lasse Sommer Kristensen, Marianne Bach Treppendahl, Mia Seremet Girkov, Fazila Asmar, Helene Myrtue Nielsen, Tina Kjeldsen, Elisabeth Ralfkiaer, Lise Lotte Hansen, Kirsten Grønbæk. Investigation of MGMT methylation patterns in diffuse large B-cell lymphoma using a novel allelic methylation-specific PCR pyrosequencing assay. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B28.

Monitoring of Lactobacillus sp. inoculated in the reactor to evaluate the solubilization efficiency of primary sludge

A new approach for the solubilization of primary sludge using the fatty-acid-producing bacteria, Lactobacillus brevis, was investigated in a laboratory-scale solubilization reactor under anaerobic conditions. Compared with a non-inoculated experiment, the 10%(v/v) inoculated experiment in an anaerobic bioreactor increased SCOD by more than 59%, and led to 50.5% COD solubilization as well as the production of mixed acids (acetic acid, lactic acid, propionic acid, butyric acid, valeric acid, etc.) after 4 days of reaction time. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA fragments revealed that the inoculated L. brevis became the predominant population in the bacterial community during the anaerobic solubilization process. PCA analysis performed on the molecular weights from the DGGE band revealed that the microbial community structure of each sample was grouped by the presence of lactic acid. This suggests that bioaugmentation of the organism might be useful for enhancing the solubilization of primary sludge. L. brevis in the solubilization reactor was monitored using 16S rDNA-based quantitative real-time PCR assays. A specific probe for the detection of L. brevis was designed and no specific PCR products were amplified, as verified by the cycle threshold value.

Identification and typing of respiratory adenoviruses in Guangzhou, Southern China using a rapid and simple method

Human adenoviruses (HAdVs), especially HAdV-B3, -E4 and -B7, are associated with Acute Respiratory Disease in Chinese children, and occasionally in adults. In order to establish and document the profiles of the respiratory adenovirus pathogen among children in Guangzhou, Southern China, a rapid, simple and practical method for identification and typing of respiratory adenoviruses was developed and evaluated. One pair of universal PCR primers was designed according to the conserved region of the hexon gene, which can detect not only HAdV-B3, -E4 and -B7, but also HAdV-B14, -F40 and -F41, with a specific 300bp PCR product. Three pairs of type-specific PCR primers were also designed according to the hypervariable regions of the hexon gene to type HAdV-B3, -E4 and -B7 by three independent PCR reactions, making it easy to optimize the PCR conditions. By using this method, one hundred throat swab specimens collected during Oct 2010 to Dec 2011 and suspected of being positive for adenoviral infection were identified and typed for adenoviruses. Of these samples, fifty-five were adenovirus-positive. The most common HAdV type was HAdV-B3, identified in 92.7% of samples, which is not only consistent with the data reported in 2004-2006, but also consistent with the recent report in Hangzhou, eastern China, indicating that HAdV-B3 has been circulating in Guangzhou, and maybe in eastern China, for many years. The method for the respiratory adenovirus identification and typing we developed is rapid, simple and practical, which has a potential in the real-time surveillance of circulating adenovirus strains and also to provide etiological evidence for the adenovirus-relative disease control and prevention in China.

Multilevel D-loop PCR identification of hunting game

The control region of mtDNA (D-loop) was used for hair samples of the five hunting game species identification: red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries musimon), and wild boar (Sus scrofa). For D-loop multilevel PCR detection scheme was applied in six primers (CE CVZV 1=5′-GATCACGAGCTTGATCACCA-3′; CE CVZV 2=5′-AGGAGTGGGCGATTTTAGGT-3′; DD CVZV 3=5′-CGCGTGAAACCAACAACCCGC-3′; DD CVZV 4=5′-CCGGGTCGGGGCCTTAGACG-3′; SSW CVZV 5=5′-ACACGTGCGTACACGCGCATA-3′; SSW CVZV 6=5′-GGTGCCTGCT T TCGTAGCACG-3′) designed to identify unknown biological samples of the hunting game animals. The PCR reaction volume was 25μl at conditions 95°C for 2min, 94°C for 30s, 60°C for 30s, 72°C for 30s, 35cycles, with last extension at 72°C for 10min. D-loop mtDNA amplicons of the game animals are characterized with specific PCR product sizes depending on species: red deer=163bp and 140bp, fallow deer=280bp and 138bp, roe deer=303bp, 280bp, 160bp and 138bp, mouflon=299bp and 178bp, wild boar=137bp and 229bp.

SERS Primers and Their Mode of Action for Pathogen DNA Detection

SERS primers have been used to directly detect specific PCR products utilizing the difference in adsorption of single-stranded and double-stranded DNA onto nanoparticle surfaces. Herein, seven parameters important for improved positive SERS assays for real applications were investigated. First, we applied a model system for optimization experiments, followed by a PCR assay to detect pathogen DNA, and then the introduction of a new assay that utilizes the 5'→3' exonuclease activity of Taq DNA polymerase to partly digest the SERS probe, generating dye-labeled single-stranded DNA increasing the SERS signals for detection of pathogen DNA. Applying the model system, it was found that uni-molecular SERS primers perform better than bi-molecular SERS primers. However, within the PCR assays, it was found that uni- and bi-molecular SERS primers performed very similarly, and the most reproducible results were obtained using the 5'→3' exonuclease digestion assay. These SERS-based assays offer new routes over conventional fluorescence-based techniques without compromising sensitivity or selectivity.

First Report of Soybean vein necrosis-associated virus Affecting Soybeans in Alabama

Soybean vein necrosis-associated virus (SVNaV) is a relatively new disease of soybeans in the United States. This is the first report of SVNaV in Alabama. The disease was confirmed to be SVNaV by ELISA and sequencing virus specific PCR products. Confirmation of the disease in Alabama is an important step in developing management recommendations for growers. Accepted for publication 10 May 2013. Published 29 July 2013.

Determining Fungi rRNA Copy Number by PCR

The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within a standard qPCR reaction. The first control developed was the internal standard control gene, benA. This gene encodes for β-tubulin and was selected based on its single-copy nature. The second control developed was the standard control plasmid, which contained a fragment of the ribosomal RNA (rRNA) gene and produced a specific PCR product. The results confirm the multicopy nature of the rRNA region in several filamentous fungi and show that we can quantify fungi of unknown genome size over a range of spore extractions by inclusion of these two standard controls. Advances in qPCR have led to extremely sensitive and quantitative methods for single-copy genes; however, it has not been well established that the rRNA can be used to quantitate fungal contamination. We report on the use of qPCR, combined with two controls, to identify and quantify indoor fungal contaminants with a greater degree of confidence than has been achieved previously. Advances in indoor environmental health have demonstrated that contamination of the built environment by the filamentous fungi has adverse impacts on the health of building occupants. This study meets the need for more accurate and reliable methods for fungal identification and quantitation in the indoor environment.

Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA

Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription. We express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction. The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15. kod-ssb may in future be used to enhance DNA and cDNA amplification.

Determination of PCR products by CE with contactless conductivity detection

The use of CE with contactless conductivity detection for the determination of PCR products is demonstrated for the first time. The separation of specific length PCR products according to their size could be achieved using 5% PVP as a sieving medium in a separation buffer consisting of 20 mM Tris and 20 mM 2-(cyclohexylamino)ethansulphonic acid (pH 8.5). A fused silica capillary of 60 cm length and 50 μm id and an applied separation voltage of -15 kV were employed and separations could be completed within 20-50 min. PCR amplified DNA fragments of different sizes obtained from different bacterial plasmid templates as well as a fragment from genomic DNA of genetically modified soybeans could be successfully identified.

Capabilities and Challenges of Examination of Gene Expression for Quality Assessment of Domestic Cat Embryos

Early embryos are characterized by an accurately controlled gene expression pattern that might be deregulated during in vitro culture (IVC). The expression pattern of the developmental genes may serve as markers for embryo quality. Here, we examined the temporal pattern of relative mRNA abundance of genes important for early embryonic development in embryos produced by different fertilization methods [in vitro fertilization (IVF) vs intracytoplasmic sperm cell injection (ICSI)] and sperm sources (fresh vs frozen-thawed) applying reverse transcriptase (RT) PCR. The temporal pattern of gene expression was found to be gene specific and similar in all four examined groups in a semi-quantitative assay. In morulae, higher relative mRNA levels were found in embryos generated with fresh sperm, whereas in blastocysts, mRNA abundance tended to be higher in embryos produced with cryopreserved sperm cells. This indicates an influence of sperm cryopreservation on the temporal gene expression pattern in early cat embryos. We also examined relative mRNA abundances by real-time quantitative RT-PCR in blastocysts. In this context, blastocysts produced with fresh semen tended to have lower DNA methyltransferase 3A (DNMT3A) but higher gap junction protein alpha 1 (GJA1) and octamer-binding transcription factor 4 (OCT4) mRNA levels compared with those derived with frozen-thawed semen. We conclude that assessing embryo quality by measuring gene expression pattern in early embryos is challenging because of a high variability between individual embryos.