ABSTRACT
Using dinucleotide repeats for carrier detection and prenatal diagnosis of haemophilia A patients, led us to find different alleles and their frequencies in Iranian population. Polymerase chain reaction (PCR) amplification of two short tandem repeat (STR) loci of factor VIII (FVIII) gene was performed, and the PCR products were resolved on 10% native polyacrylamide gel, and samples were analysed with sequenced DNA markers made of PCR cloning of the dinucleotide FVIII gene fragments. Seven different alleles were observed for intron 13 STR, having 18-24 (CA) repeating units and five alleles for intron 22 STR having 24-28 repeating units of (CACT). Bands produced during dinucleotide study were defined in detail so this could improve the genotyping of heterozygotes and homozygotes. Conformational band produced were characterized to specify the dinucleotide pattern. Our results confirm the Hardy-Weinberg proportions of the heterozygosity rate of the 85 analysed individuals. The observed heterozygosity rate for intron 13 and 22 was 52% and 59% respectively. Our data also indicate that our population is closer to caucasians than to any other populations. Finding different dinucleotide repeat alleles and their frequencies has made it possible to identify carriers and provide prenatal diagnosis with more confidence. This allows antenatal diagnosis to be performed in the vast majority of carriers.
Subject(s)
Factor VIII/genetics , Genetic Carrier Screening/methods , Hemophilia A/genetics , Repetitive Sequences, Nucleic Acid , Alleles , Base Sequence , DNA/chemistry , Female , Hemophilia A/diagnosis , Hemophilia A/economics , Humans , Introns/genetics , Iran/ethnology , Male , Pedigree , Polymerase Chain Reaction , Polymorphism, Restriction Fragment LengthABSTRACT
beta-Thalassemias are a heterogeneous group of autosomal recessive disorders, characterized by reduced or absence of the beta-globin chain production by the affected alleles. Transplantation of genetically corrected autologous hematopoietic stem cell (HSC) is an attractive approach for treatment of these disorders. Gene targeting (homologous recombination) has many desirable features for gene therapy due to its ability to target the mutant genes and restore their normal expression. In the present study, a specific gene construct for beta-globin gene replacement was constructed consisting of: two homologous stems including, upstream and downstream regions of beta-globin gene, beta-globin gene lying between hygromycin and neomycin resistant genes as positive selection markers and thymidine kinase expression cassettes at both termini as negative selection marker. All segments were subcloned into pBGGT vector. The final plasmid was checked by sequencing and named as pFBGGT. Mammalian cell line COS-7 was transfected with linear plasmid by lipofection followed by positive and negative selection. DNA of the selected cells was analyzed by PCR and sequencing to confirm the occurrence of homologous recombination. In this novel strategy gene replacement was achieved in one step and by a single construct.
Subject(s)
Gene Rearrangement/genetics , Gene Targeting/methods , Globins/genetics , Protein Engineering/methods , Recombination, Genetic/genetics , Transfection/methods , Transgenes/genetics , Animals , COS Cells , Chlorocebus aethiops , Genetic Therapy/methods , Humans , Selection, Genetic , beta-Thalassemia/genetics , beta-Thalassemia/therapyABSTRACT
Recent investigations have shown that nuclear magnetic resonance (NMR) can be used in conjunction with a suitable chemical dosimeter to estimate the dose from ionizing radiation (Gore et al., Phys Med. Biol. 29, 1189-1197, 1984). Based on this fact it was proposed that spatial dose distributions can be measured in gels infused with the chemical dosimeter using NMR imaging. There have been few such attempts and they provided only qualitative results. In this paper, we report results demonstrating the feasibility of obtaining quantitative dose distribution measurements by this technique. It is shown that quantitative dose distribution measurements necessitate the calculation of relaxation rate maps. We have determined that the spin-spin relaxation rate is a more sensitive parameter than the spin-lattice relaxation rate. It is also demonstrated that the addition of chemical sensitizers could improve the dose sensitivity of the measured NMR parameters. The two features characterizing a photon beam, depth-dose relationship, and beam profile as measured by this technique are in good agreement with the measurements using conventional methods, ionization chambers, and film dosimetry.
