Agarose Gel Separation Research Articles

Clinical value assessment for serum hsa_tsr013526 in the diagnosis of gastric carcinoma.

Gastric carcinoma (GC) is a malignant tumor that is detrimental to human health. Transfer RNA-derived small RNAs are a newly identified class of noncoding small RNAs with specific biological functions that are aberrantly expressed in cancer. The aim of this study was to investigate the potential of hsa_tsr013526 as a biomarker for GC. Quantitative real-time fluorescence polymerase chain reaction was used to detect the expression level of hsa_tsr013526. The molecular characteristics of hsa_tsr013526 were verified by agarose gel electrophoresis, Sanger sequencing, and separation of nuclear and cytoplasmic RNA fractions. By testing the receiver operating characteristic (ROC) curves, the diagnostic efficiency of GC using hsa_tsr013526 was determined. Finally, we predicted the downstream of hsa_tsr013526 using functional assays and bioinformatics analysis. Serum expression of hsa_tsr013526 was higher in GC patients than in healthy donors. Serum expression showed differential changes in GC patients, gastritis patients, and healthy donors. Chi-squared tests showed that high expression of hsa_tsr013526 was significantly correlated with T stage, lymphatic metastasis, and tumor node metastasis stage. ROC curve analysis indicated that GC patients could be discriminated from healthy donors or gastritis patients based on their serum levels of hsa_tsr013526. Furthermore, hsa_tsr013526 expression was significantly reduced in postoperative GC patients (p = .0016). High expression of hsa_tsr013526 promotes gastric cancer cell proliferation, invasion, and migration. Serum hsa_tsr013526 was stable and specific, and could be used for dynamic monitoring of GC patients. Therefore, hsa_tsr013526 may be a new biomarker for the diagnosis and postoperative monitoring of GC patients.

Identification of polymorphism in APAF1 gene in holstein cattle

The article presents the data of DNA testing of Holstein cattle by the APAF1 gene. The structure of the Tatarstan population was studied in comparison with world experience. The aim of the study was to study the allelic polymorphism of the apoptosis peptide activating factor 1 gene (APAF1; g.C6315040T; p.Gln579Q→X), the cause of the fertility haplotype (HH1), in the domestic population of Holstein cattle of the Republic of Tatarstan. Genotyping was carried out by PCR-RFLP followed by electrophoretic separation in agarose gel in the presence of ethidium bromide. For the first time in the Republic of Tatarstan in the conditions of the Dairy farm "named Lenin” carried out the identification of cattle by the locus of APAF1 - BstC8 I, assessed the genetic equilibrium and structure of the population. As a result of gene diagnostics, two alleles and three genotypes were identified. The frequency of occurrence of alleles Q and X was 0.988 and 0.012; genotypes QQ and QX – 97.5 and 2.5%, respectively. Since homozygous XX embryos do not survive, they are never found among born animals. Chi-quad testing showed that the studied population is in genetic equilibrium according to the Hardy-Weinberg law. Our studies confirm an insignificant share of the presence of animals-carriers of the lethal allele in the population of Holstein cattle of domestic selection of the Republic of Tatarstan. In order to contain the spread of the mutant allele X of the apoptosis peptide activating factor 1 gene, it is recommended to conduct DNA testing of polymorphism of the APAF1 gene during the selection of pairs for breeding.

Development of Wild Boar Species-Specific DNA Markers for a Potential Quality Control and Traceability Method in Meat Products

In the food supply chain, quality control has a very important role in maintaining customer confidence. In the EU, food safety aspects are strictly regulated; however, composition requirements and standard control methods are generally undefined. The rapidly increasing wild boar population has a growing market share in venison or game meat production. Several methods have been described for species identification and control of composition in food products, but only some of these are suitable for routine measurements. The aim of our research was to design a rapid, reliable and simple PCR insertion/deletion (InDel)-based genetic tool suitable for species identification in food quality control laboratories. In total, 59 different swine (Sus scrofa) whole genomes were tested with bioinformatic tools to identify wild boar-specific insertions or deletions. Three independent InDels were suitable for marker development, multiplex PCR amplification and separation in agarose gel. Altogether, 209 samples of wild boar and ten other domestic pig breeds were taken for DNA extraction and validation of the three multiplexed InDel markers. Statistical analysis showed a very high combined predictive value (0.996), indicating the capability of the newly developed markers to detect wild boars with a probability over 99%. Breed assignment tests confirm that the InDel markers developed are suitable for rapid, sensitive and reliable identification of the wild boar meat content of food products. The use of the reported method in food quality control can mean a simple and cost-effective way to maintain consumer confidence and to support the competitiveness of fair producers.

A novel myelin basic protein transcript variant in the murine central nervous system.

Myelin basic protein is a multifunctional protein whose primary role is to adhere membranes of the myelin sheath. There are various isoforms that have been identified, 6 distinct isoforms in human and 13 distinct isoforms in mice. These distinct isoforms are the product of alternative splicing of a single gene. The present study sought out to identify the different isoforms found in the murine central nervous system. Neuronal tissue (brain) from five different C57BL6/J mice at 2 months of age was harvested and used for mRNA extraction. mRNA was reversed transcribed to cDNA and transcripts were detected through PCR amplification and DNA agarose gel separation. Primers for exon 1, exon 5b and exon 11 of the myelin basic protein gene were used to capture all the possible transcripts that are naturally found in the murine central nervous system. Unknown transcript was sequenced at Genewiz facilities (South Plainfield, NJ) and mass spectrometry protein sequence analysis demonstrated the presence of a novel myelin basic protein transcript variant. We identified a novel transcript variant of myelin basic protein. This novel transcript variant corresponds to a myelin basic protein of 32.5 kDa which has not been previously reported. This novel transcript variant presents relevant clinical significance to various demyelinating diseases due to its contribution to the understanding of the natural state of the murine central nervous system.

Discovery, Characterization, and Linkage Mapping of Simple Sequence Repeat Markers In Hazelnut

From the genome sequence of hazelnut (Corylus avellana), 192 new polymorphic simple sequence repeat (SSR) markers were developed, characterized, and used to investigate genetic diversity in 50 accessions. Next-generation sequencing allows inexpensive sequencing of plant genomes and transcriptomes, and efficient development of polymorphic SSR markers, also known as microsatellite markers, at low cost. A search of the genome sequence of ‘Jefferson’ hazelnut identified 9094 fragments with long repeat motifs of 4, 5, or 6 base pairs (bp), from which polymorphic SSR markers were developed. The repeat regions in the ‘Jefferson’ genome were used as references to which genomic sequence reads of seven additional cultivars were aligned in silico. Visual inspection for variation in repeat number among the aligned reads identified 246 as polymorphic, for which primer pairs were designed. Polymerase chain reaction (PCR) amplification followed by agarose gel separation indicated polymorphism at 195 loci, for which fluorescent forward primers were used to amplify the DNA of 50 hazelnut accessions. Amplicons were post-PCR multiplexed for capillary electrophoresis, and allele sizes were determined for 50 accessions. After eliminating three, 192 were confirmed as polymorphic, and 169 showed only one or two alleles in each of the 50 cultivars, as expected in a diploid. At these 169 SSRs, a total of 843 alleles were found, for an average of 4.99 and a range of 2 to 17 alleles per locus. The mean observed heterozygosity, expected heterozygosity, polymorphism information content, and the frequency of null alleles were 0.51, 0.53, 0.47, and 0.03, respectively. An additional 25 primer pairs produced more than two bands in some accessions with an average of 6.8 alleles. The UPGMA dendrogram revealed a wide genetic diversity and clustered the 50 accessions according to their geographic origin. Of the new SSRs, 132 loci were placed on the linkage map. These new markers will be useful for diversity and parentage studies, cultivar fingerprinting, marker-assisted selection, and aligning the linkage map with scaffolds of the genome sequence.

An efficient method for identifying Escherichia coli strains isolated from various (Gallus gallus domesticus) organs

Tracing transmission routes and identifying pathogen sources are important issues in preventive measures aimed at controlling human and animal infectious diseases. A fast and accurate method for bacterial strain identification (genotyping) allows a scientifically sound planning of preventive schemes. Despite the existence of numerous bacteri a genotyping techniques, there is still room for developing a unified typing approach that would be applicable to a variety of bacterial species. The aim is to develop a genotyping method allowing identification of E. coli strains circulating at Russian chicken farms. The method is based on the earlier proposed idea of double digestion and selective labeling of DNA restriction fragments (DDSL). Bacterial genomic DNA is simultaneously digested with two restriction enzymes and labeled with biotinylated deoxynucleoside triphosphates with the presence of DNA polymerase. The enzymes are chosen in silico for each bacterial species so that a limited number of DNA fragments are generated for subsequent separation in conventional agarose gel. After implementation of the study with E. coli isolates, adequate reproducibility and a high discriminatory power of the technique were demonstrated. This approach was previously applied to genotyping other pathogenic bacterial species. The advantages of the proposed technique are the short turnaround time of analysis and easy availability of reagents and equipment. Transmission of a pathogen among chicken within one farm and existence of slightly different E. coli genotypes in various organs of the same individual were observed. Bacterial isolates obtained from any organ except the intestine were suitable for genotyping. Chicken intestine contains endogenous E. coli strains, which hamper the interpretation of the genotyping data obtained for a set of isolates. Thus, our work demonstrates the potential of the DDSL method for genotyping field E. coli isolates in the context of molecular epizootology.

Efficient method for identification of Escherichia coli strains isolated from various chicken (Gallus gallus domesticus) organs

Tracing of transmission routes and identification of pathogen sources are important issues in preventive measures aimed at controlling human and animal infectious diseases. A fast and accurate method for bacterial strain identification (genotyping) allows scientifically sound planning of preventive schemes. Despite the existence of numerous bacterium genotyping techniques, there is still room for developing a unified typing approach that would be applicable to a variety of bacterial species. The aim is to develop a genotyping method allowing identification of E. coli strains circulating at Russian chicken farms. The method is based on the earlier proposed idea of double digestion and selective labeling of DNA restriction fragments (DDSL). Bacterial genomic DNA is simultaneously digested with two restriction enzymes and labeled with biotinylated deoxynucleoside triphosphates with the presence of DNA polymerase. The enzymes are chosen in silico for each bacterial species so that a limited number of DNA fragments be generated for subsequent separation in conventional agarose gel. After implementation of the study with E. coli isolates, adequate reproducibility and high discriminatory power of the technique were demonstrated. This approach was previously applied to genotyping other pathogenic bacterial species. The advantages of the proposed technique are the short turn-around time of analysis and easy availability of reagents and equipment. Transmission of a pathogen among chicken within one farm and existence of slightly different E. coli genotypes in various organs of the same individual were observed. Bacterial isolates obtained from any organ except the intestine were suitable for genotyping. Chicken intestine contains endogenous E. coli strains, which hamper the interpretation of genotyping data obtained for a set of isolates. Thus, our work demonstrates the potential of the DDSL method for genotyping field E. coli isolates in the context of molecular epizootology.

Hypertonic conditions enhance cartilage formation in scaffold-free primary chondrocyte cultures.

The potential of hypertonic conditions at in vivo levels to promote cartilage extracellular matrix accumulation in scaffold-free primary chondrocyte cultures was investigated. Six million bovine primary chondrocytes were cultured in transwell inserts in low glucose (LG), high glucose (HG), or hypertonic high glucose (HHG) DMEM supplemented with fetal bovine serum, antibiotics, and ascorbate under 5 % or 20 % O2 tension with and without transforming growth factor (TGF)-β3 for 6 weeks. Samples were collected for histological staining of proteoglycans (PGs) and type II collagen, analysis by quantitative reverse transcription plus the polymerase chain reaction (RT-PCR) of mRNA expression of aggrecan and procollagen α1 (II) and of Sox9 and procollagen α2 (I), and quantitation of PGs and PG separation in agarose gels. Cartilage tissues produced at 20 % O2 tension were larger than those formed at 5 % O2 tension. Compared with LG, the tissues grew to larger sizes in HG or HHG medium. Histological staining showed the strongest PG and type II collagen staining in cartilage generated in HG or HHG medium at 20 % O2 tension. Quantitative RT-PCR results indicated significantly higher expression of procollagen α1 (II) mRNA in cartilage generated in HHG medium at 20 % O2 tension compared with that in the other samples. TGF-β3 supplements in the culture medium provided no advantage for cartilage formation. Thus, HHG medium used at 20 % O2 tension is the most beneficial combination of the tested culture conditions for scaffold-free cartilage production in vitro and should improve cell culture for research into cartilage repair or tissue engineering.

Association of aldosterone synthase (CYP11B2) gene −344T/C polymorphism with the risk of primary chronic glomerulonephritis in the Polish population

We evaluate whether angiotensinogen AGT M235T (rs699), angiotensin-converting enzyme ACE (I/D) (rs4646994) and aldosterone synthase CYP11B2 -344C/T (rs1799998) polymorphisms can be genetic risk factors of chronic glomerulonephritis (GN) in the Polish population. The study was conducted in 140 patients with primary chronic GN: mesangial proliferative GN (MesPGN) (n = 49), IgA nephropathy (IgAN) (n = 31), membranous nephropathy (MN) (n = 27), focal segmental glomerulosclerosis (FSGS) (n = 25), membranoproliferative GN (MPGN) (n = 4), and minimal change disease (MCD) (n = 4), and controls (n = 187). Genotypes were determined by HRM curve analysis for AGT M235T, by PCR and agarose gel separation for ACE (I/D), and by PCR-RFLP for CYP11B2 -344C/T. We found a significant association of the CYP11B2 -344C/T polymorphism in the recessive model with all subtypes of GN (OR = 1.925 (95% CI = 1.152-3.219, p = 0.0118, p(corr) = 0.0354)). We also observed that the CYP11B2 -344C/T polymorphism in the recessive model may also be an independent significant risk factor of IgAN (OR = 2.743 (95% CI = 1.219-6.172, p = 0.0122, p(corr) = 0.0366)), FSGS (OR = 2.895 (95% CI = 1.200-6.985, p = 0.0145, p(corr) = 0.0435)), and all proliferative GNs (MesPGN, IgAN, MPGN) (OR = 2.171 (95% CI = 1.211-3.894, p = 0.0084, p(corr) = 0.0252)). Our results suggest that the CYP11B2 -344C/T polymorphism might be an independent risk factor of IgAN, FSGS and all proliferative chronic GNs.

Development and mapping of SNP assays in allotetraploid cotton

A narrow germplasm base and a complex allotetraploid genome have made the discovery of single nucleotide polymorphism (SNP) markers difficult in cotton (Gossypium hirsutum). To generate sequence for SNP discovery, we conducted a genome reduction experiment (EcoRI, BafI double digest, followed by adapter ligation, biotin–streptavidin purification, and agarose gel separation) on two accessions of G. hirsutum and two accessions of G. barbadense. From the genome reduction experiment, a total of 2.04 million genomic sequence reads were assembled into contigs with an N50 of 508 bp and analyzed for SNPs. A previously generated assembly of expressed sequence tags (ESTs) provided an additional source for SNP discovery. Using highly conservative parameters (minimum coverage of 8× at each SNP and 20% minor allele frequency), a total of 11,834 and 1,679 non-genic SNPs were identified between accessions of G. hirsutum and G. barbadense in genome reduction assemblies, respectively. An additional 4,327 genic SNPs were also identified between accessions of G. hirsutum in the EST assembly. KBioscience KASPar assays were designed for a portion of the intra-specific G. hirsutum SNPs. From 704 non-genic and 348 genic markers developed, a total of 367 (267 non-genic, 100 genic) mapped in a segregating F2 population (Acala Maxxa × TX2094) using the Fluidigm EP1 system. A G. hirsutum genetic linkage map of 1,688 cM was constructed based entirely on these new SNP markers. Of the genic-based SNPs, we were able to identify within which genome (‘A’ or ‘D’) each SNP resided using diploid species sequence data. Genetic maps generated by these newly identified markers are being used to locate quantitative, economically important regions within the cotton genome.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-011-1780-8) contains supplementary material, which is available to authorized users.

Functional characterization of watermelon ( Citrullus lanatus L.) EST–SSR by gel electrophoresis and high resolution melting analysis

The present work was conducted to characterize the functionality of 257 watermelon EST–SSR primer pairs for their PCR amplification and polymorphisms. EST–SSR markers were tested on DNA sample panels of six watermelon cultigens and two related species of Citrullus lanatus var. citroides and Citrullus colocynthis based on agarose gel electrophoresis and high resolution melting (HRM) analysis. Successful PCRs were shown for 240 primer pairs (79%), and 173 primer pairs (67%) were polymorphic in a watermelon DNA sample panel on agarose gel electrophoresis. In addition, HRM analysis of 24 EST–SSR markers that were monomorphic on agarose gel separation identified an additional 19 polymorphic markers, indicating that HRM is an efficient tool for the rapid screening of sequence variations and allele discrimination. In the assessment of genetic relationships, six watermelon cultivars were closely related together (0.91–0.97) and demonstrated a narrow genetic base in the watermelon genetic pool. A high level of genetic dissimilarity (0.36–0.97) was shown between watermelon species and other related species. Marker transferability to melon species ( Cucumis melo L.) was examined by cross-species PCR amplification and genetic diversity assessment in eight melon cultigens. Of the 257 EST–SSR primer pairs, 79 (32.9%) showed successful PCR amplification from melon DNA samples. A dendrogram of the genetic relationship based on 22 EST–SSR markers showed a clear classification of melon genotypes in accordance to fruit characteristics. The EST–SSR markers characterized in this study will contribute to diverse genomic investigations and breeding efforts, including comparative genome mapping, marker-assisted selection, and DNA fingerprinting for genetic diversity and cultivar identification in many cucurbit crops.

Validation of ten new polymorphic tandem repeat loci and application to the MLVA typing of Burkholderia pseudomallei isolates collected in Singapore from 1988 to 2004

Multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) has been shown to be very promising for the typing of Burkholderia pseudomallei and mallei. The currently available set of loci requires high resolution allele size measurement due to short repeat units. The present work was aimed at expanding the available set of VNTR loci, and generating data from a collection of 102 B. pseudomallei strains isolated in Singapore between 1988 and 2004 including few additional strains of various origins as references. Ten new VNTRs with a longer array size have been identified compatible with standard agarose gel separation, and a reference database of 72 genotypes was created which can be queried on the Internet.

Genetic polymorphism of an alpha 1-protease inhibitor in mink plasma.

Two-dimensional electrophoretic analysis of mink (Mustela vison Schreber) plasma proteins was done by a first-dimension separation in agarose gel (pH 8.6), followed by a second dimension in horizontal polyacrylamide gel (pH 9.0). Two α1-globulins, designated Pi-1 and Pi-2, and another protein, designated Pi-3 were found to inhibit the esterolytic activity of bovine trypsin and bovine chymotrypsin. The Pi-1 segregation analysis in limited family data supported the hypothesis that the three Pi-1 types observed were controlled by two codominant, autosomal alleles. Each of the Pi-1 homozygote types showed one strong, broad fraction while the Pi-1 heterozygote type had two fractions. The electrophoretic mobility and patterns of Pi-1 indicated that this protein was most probably identical to the polymorphic post albumin (Pa) in mink plasma, reported by Saison in 1968.

Two-dimensional gel electrophoresis of dog plasma proteins: genetic polymorphism of an alpha 1-protease inhibitor and another postalbumin.

Two-dimensional electrophoretic analysis of dog plasma proteins was done by a first-dimension separation in agarose gel (pH 5.4) followed by a second-dimension separation in horizontal poly-acrylamide gel (pH 9.0). Two groups of α1-globulins, designated Pi-1 and Pi-2, were found to be protease inhibitors. Extensive genetic polymorphism of Pi-1 and of another unidentified α-globulin, tentatively designated postalbumin (Pa), was observed. Family data supported the hypothesis that Pi-1 and Pa polymorphisms were controlled by codominant, autosomal alleles. Three Pi-1 alleles and two Pa alleles were observed in 60 breeds (1119 dogs) studied. Each of the Pi-1 alleles gave rise to a single strong fraction while each of the Pa alleles showed a major and a minor anodic fraction. The present study, along with earlier reports, showed that some of the proteins and enzymes (albumin, transferrin, eserine resistant esterase, α1-protease inhibitor and postalbumin) in plasma exhibit considerably more polymorphism than that reported for hemoglobin and some of the red cell and tissue enzymes of the domestic dog.

Application and evaluation of double digest selective label (DDSL) typing technique for Pseudomonas aeruginosa hospital isolates

This study describes the application and evaluation of a recently developed fast bacterial typing technique (double digest selective label — DDSL) for hospital isolates of Pseudomonas aeruginosa. The protocol was based on a simultaneous double digestion/labelling reaction which was performed in a single reaction tube. After agarose gel separation selectively tagged restriction fragments were transferred using deonised water to a nylon membrane and visualized by a colour reaction. Starting from overnight culture, turn around time using this technique was only 8 h. The DDSL typing technique was applied for 77 hospital isolates. Among them 63 isolates were also typed by PFGE and the typing results were compared with those of DDSL. In conclusion, both techniques discriminated bacterial isolates into the same major clusters. DDSL proved to be as discriminatory as PFGE but much faster and easier to set up in a standard microbiological laboratory.

Evaluation of Genetic Variability in Kenkatha Cattle by Microsatellite Markers

Kenkatha cattle, a draft purpose breed, which can survive in a harsh environment on low quality forage, was explored genetically exploiting FAO-suggested microsatellite markers. The microsatellite genotypes were derived by means of the polymerase chain reaction (PCR) followed by electrophoretic separation in agarose gels. The PCR amplicons were visualized by silver staining. The allelic as well as genotypic frequencies, heterozygosities and gene diversity were estimated using standard techniques. A total of 125 alleles was distinguished by the 21 microsatellite markers investigated. All the microsatellites were highly polymorphic with mean allelic number of 5.95±1.9 (ranging from 3-10 per locus). The observed heterozygosity in the population ranged between 0.250 and 0.826 with a mean of 0.540±0.171, signifying considerable genetic variation. Bottleneck was examined assuming all three mutation models which showed that the population has not experienced bottleneck in recent past. The population displayed a heterozygote deficit of 21.4%. The study suggests that the breed needs to be conserved by providing purebred animals in the breeding tract.

History, genetic differentiation and conservation strategies for disjunct populations of Sibiraea species from Southeastern Europe and Asia

The genetic structure of Croatian Sibirea (Sibiraea croatica), a rare and endemic tertiary relic of Croatian and Herzegovinian flora, and its relationship with sibirea from Southern Russia and Southern Siberia (Sibiraea altaiensis) was studied using amplification, restriction and sequencing of the ITS region in genomic DNA and cpDNA and their comparisons with sequences of the Rosaceae species obtained from GenBank. The restriction analysis and separation in agarose gel showed no differences in length of the digested cpDNA between or within populations. Sequencing showed only minor variability between populations. Only a minor difference of 6 bp duplication in DNA amplified with ccmp 10-R and trnM primer pair was noticed in two geographically distinct populations. No differences in the restriction pattern for the ITS region in genomic rDNA indicates that all samples of sibirea belong to the same species since the ITS region was proven to be conserved within one taxonomic species. The minor differences that were␣obtained support the hypothesis that sibirea is an old tertiary relic that shows a minor variability, confirming previous preliminary results from comparisons of the Croatian and Altaic sibireas at the morphological level. Our data suggests that Croatian sibirea from the Balkan is a disjunct population identical to the Altaic species. Due to its disjunct occurrence in Southeastern Europe, the endemic status in the Dinarics, a relic that survived the glaciations, it deserves active conservation approaches through support of traditional use of high-mountain pastures for reducing natural reforestation of sibirea ancient sites.

178. Site-Specific Base Alteration in the Promoter Region of the Human Gamma Globin Gene by Single-Stranded Oligonucleotides for Gene Therapy of Sickle Cell Disease

Top of pageAbstract The human –globin chain has shown greater capacities for inhibiting the polymerization of sickle (S) hemoglobin (Hb) than the –globin chain itself. Moreover, increasing the synthesis of fetal (F) Hb in adults by specific pharmacologic agents has been developed as an alternative strategy to ameliorate sickle cell anemia and –thalassemia. Unfortunately, their clinical application is hampered by their considerable toxicity. Some naturally occurring point mutations in the promoters of A– and G– globin genes are associated with increased HbF in adult erythroid cells. In particular, the C to G mutation at –202 in the G– gene promoter increases the level of HbF up to 15–25% of total hemoglobin, which is sufficient for ameliorating the clinical manifestations of sickle cell anemia. Thus, reactivation of –globin gene expression by targeted mutagenesis of specific sites provides a novel strategy for gene therapy of sickle cell disease. Short (45–100 nt) single-stranded oligonucleotides (SSOs) have been shown to mediate site-specific alteration of single base pairs in both episomal and chromosomal target genes in mammalian cells. The exchange reaction can result in either repair of a lesion or creation of a mutation and is mediated through endogenous DNA repair pathway(s). The aim of this study was to determine if SSO directed DNA repair could introduce specific single base changes in the G– gene promoter region. Forty-five mer SSOs were designed homologous to the G– globin promoter target site –202 except for the middle base which was designed to induce a C to G mutation. This single base alteration at nt –202 also changes the wild-type (wt) Ban II restriction endonuclease site to an Ehe I site, providing a restriction fragment length polymorphism (RFLP) for detection of the desired C to G change. To increase resistance to intracellular nucleases, the SSOs were synthesized with either amine or phosphorothioate linkages at both ends. At various times following transfection of human cell lines with the modified SSOs, genomic DNA was isolated from transfected and control cells and subjected to Ban II digestion. After agarose gel separation of the digested DNA, the genomic DNA from 2.3–3 kb was excised, purified and used as the template for PCR amplification of the G– promoter region spanning the target site. Only cells treated with the SSOs exhibited the desired site-specific C to G change detected by PCR-RFLP. DNA sequencing confirmed that this was the only nt change introduced in the region targeted by the SSO. Moreover, we developed a sensitive and reliable detection system which is suitable for detection of >0.1% point mutations in a wt background. Finally, our methodology for detection indicates that the C to G change was not a PCR artifact. In particular, electrophoretic separation of the digested genomic DNA was done prior to the PCR amplification for the RFLP analysis. In conclusion, 45 mer end-modified SSOs can be used to generate site-specific point mutations in the G– globin gene promoter region to specifically increase HbF expression in adult erythroid cells. This novel strategy provides a gene therapy approach for sickle cell disease and other hemoglobinopathies, independent of correcting the primary genetic lesion in the Hb gene.

Subtracted restriction fingerprinting – a new typing technique using magnetic capture of tagged restriction fragments

Molecular typing of bacterial pathogens is an important issue in the epidemiological analysis of emerging infections in humans and animals. Numerous methods have been developed for and applied to a wide variety of bacteria of medical, veterinary and zoonotic importance. The present minireview provides a description of a new typing approach designated subtracted restriction fingerprinting (SRF), its use for typing of Salmonella isolates and a comparison with the most widely used typing techniques for these bacteria. SRF is based on double restriction endonuclease digestion of whole cell DNA, followed by a fill-in reaction with specifically tagged nucleotides and subtractive capture of selected restriction fragments. This results in a reduced number of fragments optimal for separation in standard agarose gels.

IL-18 promoter polymorphisms correlate with the development of post-injury sepsis

Background. Interleukin (IL)-18 is a proinflammatory cytokine involved in the regulation of cell-mediated and innate immune responses to infection, trauma, and inflammation. Elevated levels of IL-18 have been noted to correlate with organ dysfunction after injury. This study evaluates the relationship between IL-18 promoter polymorphisms and the development of sepsis after injury. Methods. DNA was extracted from peripheral leukocytes of trauma patients with an injury severity score of 16 or greater. Patient clinical course was followed for the development of sepsis as an endpoint. Two SNPs (−607bp and −137bp) were amplified using polymerase chain reaction. Alleles were identified via agarose gel separation. Genotypes were then determined and correlated with patient data. Postinjury IL-18 levels were determined by enzyme-linked immunoassay. Results. Sixty-six patients were evaluated; 36 (52%) developed sepsis. Each SNP had 2 alleles and 3 genotypes. The SNP at −607bp had an allelic frequency of 59% for C and 41% for A; whereas −137bp was a G 79% of the time and a C 21% of the time. Individually, each SNP had no direct correlation between the patient's genotype and development of infection. However, when the −607bp CA genotype was combined with the −137bp GC genotype (CA/GC), only 4 patients (27%) developed sepsis (P =.02). Conclusions. This study supports the conclusion that IL-18 genetic promoter polymorphisms correlate with the development of postinjury sepsis. Further investigation is needed to identify the impact of variation in genotype across a range of genes involved in connected regulatory pathways. (Surgery 2003;134:351-6.)