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Volume 56, Issue 4 (7-1998)
Back in 1957, when investigators produced a triple-stranded form of DNA while studying synthetic nucleic acids, few researchers paid much attention to the discovery. However, triplex DNA was never entirely forgotton and especially since 1987 its structural and functional importance in biological systems as well as its medical applications and therapeutic potentional have been extensively studied. It was suggested that in triplex DNA, the third strand was hydrogen bonded and positioned in the major groove of the Watson-Crick duplex. Protein binding assays show that triplex formation by HR21ap inhibits Sp1 binding to the Ha-ras promoter. These results suggest that the triplex formation by the Ha-ras promoter targed oligonucleotide may provide a means to specifically inhibit transcription of this oncogene in vivo. Triplex DNA can disrupt gene transcriptions and can be used as of this oncogene in vivo. Triplex DNA can disrupt gene transcriptions and can be used as a new strategy for treating viral diseases, such as AIDS, by blocking virus reproduction. As discussed in this article, for a number of reasons, interest in oligonucleotide designed for triplex helices on dsDNA is being steadily increased (including their potential artificial repressors of gene expression, mediator of site specific DNA cleavage and therapeutic use for genetic diseases, cancer and diseases caused by viruses).
Volume 56, Issue 5 (7-1998)
Macrophage colony stimulating factor (M-CSF) has previously been shown to affect the differentiation of cells of the mono-nuclear phagocytic line. More recent studies indicate that M-CSF may have a role in pregnancy. In the present study, the expression of M-CSF in the human placenta was demonstrated. Placental mRNA was isolated and used as template for synthesis of complementary DNA (cDNA). The presence of M-CSF related sequences in the cDNA was shown by PCR and RT-PCR reactions in which M-CSF specific primers were used. In addition, it was shown that a 2.4 kb cDNA after electrophoresis and transfer to a nylon filter, hybridized with a digoxygenin labeled M-CSF specific probe.
Volume 65, Issue 1 (3-2008)
Background: Malaria is still one of the main health problems in south and southeast provinces of Iran and recently on average 10,000-30,000 malaria cases were reported annually. Mosquitoes of Anopheles superpictus are one of the main malaria vectors in Iran and have been reported from all areas of the country including central plateau and plains of Alborz and Zagrous Mountains chains, and with low numbers in shore plains of the Persian Gulf and Caspian Sea. There are variations in larval and adult morphological characters and also in vectorial capacity of this species in different areas of Iran.
Methods: This study has been conducted to investigate rate of mtDNA variation among various populations of this species in Iran. The sequence variation of an 1512 bp length of mitochondrial DNA (mtDNA) cytochrome oxidase subunits 1 and 2 (COI-COII) and an 708 bp sequences of COI gene were analyzed by PCR-RFLP and PCR-direct sequencing respectively.
Results: This study showed that there are considerable variations between and within populations. Rate of variation was 12.3 % between populations and this was 2-5% for within Baluchistan population. Totally 4 haplotypes were observed between populations where 3 occur in Baluchistan and one in other places.
Conclusion: This is the first report on existence of various haplotypes in An. superpictus in science, and presumably this species comprising siblings and is a species complex. Further studies need to confirm this result and to determine the relationship between mtDNA haplotypes and their role in malaria transmission in each locality.
Volume 65, Issue 8 (11-2007)
Background: Anopheles superpictus is one of the main malaria vectors in Iran. The mosquitoes of this species are found throughout the Iranian plateau up to 2000 meters above sea level in the Alborz Mountains, south of the Zagros Mountains, and in the plains near the Caspian Sea and Persian Gulf. It has been reported that different geographical populations of An. superpictus play different roles in malaria transmission. Based on the presence or absence of a black spot/band on the apical segment of the female maxillary palpi, two morphological forms have been reported in this species. This work has been conducted to study other morphological features as well as the genetic structure of these two forms of An. superpictus in Iran.
Methods: The different morphological characteristics of 35 different populations were observed and recorded. An 887 bp portion of the mitochondrial DNA (mtDNA) cytochrome oxidase subunit I (COI) was amplified and assayed by restriction fragment length polymorphism (RFLP) using 18 enzymes and PCR-direct sequencing techniques.
Results: Among the morphological characteristics studied, there are significant differences between the two forms with regard to the length of the palp light band (p<0.01), wing length (p<0.5), and the distance from the branching point of the II/IV veins to the tip of the wing (p<0.05). Results also revealed that these two forms are sympatric in most localities of Iran. RFLP analysis and sequences of about 710 bp of the gene showed that there was great variation between and/or within the populations, but these variations were not associated with the morphological forms.
Conclusion: This is the first comprehensive study on the morphological and molecular characteristics of An. superpictus in the literature. To determine the role of these morphological forms or genetic haplotypes in malaria transmission, further molecular, cytological, morphological, and epidemiological studies are necessary.
Volume 67, Issue 1 (4-2009)
Background: Anti-dsDNA antibodies frequently found in the sera Systemic Lupus Erythematosus patients, particularly in active disease stage. Nowadays exploit different eukaryotic and prokaryotic dsDNA as antigen source and different reagents as binder. The aim of this study to compared two dsDNA different sources and tow different kinds of reagents for binder in ELISA test.
Methods: In this study bacterial genomic DNA from E.coli (ATCC 25922) and genomic DNA from calf thymus extracted with high purity and were used as antigens for IgG anti-dsDNA detection by ELISA. To coat dsDNA in microtiter wells, tow different kinds of reagents including methylated -BSA and poly-l-lysine (for pre-coating) are used. Sera from systemic lupus erythematosus patients and from normal blood donors are used to assess sensitivity and specificity of our ELISA test in compared with IF test and commercial kits.
Results: Our results displayed pre-coating of microtiter plates with methylated -BSA reduce nonspecific binding reaction and the relative sensitivity and specificity of ELISA increased when calf thymus DNA is employed as antigenic source in compared with IF test and commercial kits 80%, 88% and 100%, 98% respectively, but when E.coli DNA is used 73%, 69% and 85%, 79%, respectively.
Conclusion: The genomic DNA from calf thymus is a potentially useful source of antigen for detection of anti-dsDNA by ELISA. Also the use of methylatted- BSA could have an effective role in reducing of nonspecific binding reactions.
Volume 67, Issue 5 (8-2009)
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MicrosoftInternetExplorer4 Background: Occult hepatitis
B infection (OBI)
is a form of hepatitis in which despite absence of detectable HBsAg, HBV-DNA
is present in peripheral blood of patients. The responsible mechanisms for
progression of OBI yet to be clarified, but some investigators believed that the genetics and
immunological parameters are different in resistant individuals and patients.
Vitamin D3 and its receptor interaction
could be involved in anti-viral immune response. The aim of this study was to
investigate the association between polymorphisms in intron 8 of VDR with OBI.
Methods: In this experimental study, the plasma samples of 3700
blood donors were collected and tested for HBsAg and anti-HBs by ELISA.
The HBsAg negative and anti-HBc positive
samples were selected and screened for HBV-DNA
using PCR. HBV-DNA positive samples were assigned as OBI cases and PCR-RFLP
was performed to examine the polymorphisms in intron 8 of VDR genes.
Results: Results of current study indicated that 352 (9.5%)
of 3700 blood samples were HBsAg- and anti-HBc+. HBV-DNA
was detected in 57/352 (16.1%) of HBsAg- and anti-HBc+ samples.
Our results showed that no significant difference was observed in Apa-1 polymorphisms
of intron 8 of VDR and OBI patients.
Conclusion: Our results demonstrated
that there are not any association between Apa-1 detected alleles and OBI, hence, it can be concluded that these alleles are
not associated with OBI and other researchers
should evaluate relation between other polymorphisms of VDR with OBI.
Volume 69, Issue 2 (5-2011)
Background: Molecular DNA markers are one of the most important tools in molecular biology labs. The size of DNA molecules is determined by comparing them with known bands of markers during gel electrophoresis. There are many different protocols to produce these kinds of molecular markers. In this study we have suggested an efficient strategy to produce molecular weight markers in industrial proportions. Methods: To achieve the desired sizes of DNA fragments, a combination of two previously known methods, restriction enzyme digestion and polymerase chain reaction (PCR), were used. The enzymatic digestion process was based on designing and constructing plasmids which equaled in size with the desired length of DNA fragments and produced the desired DNA fragment upon linearization. In the PCR method, the desired length of DNA fragments were cloned in multiple cloning sites of pTZ57R plasmid and in a PCR reaction, the new constructed plasmid was used as a template to produce the final fragment. Results: Upon application of this strategy, 2000 and 3000 bp DNA fragments were produced by enzymatic digestion of plasmids of the same size. Moreover, 100 to 1500 bp fragments were produced during PCR using only a set of forward and reverse primers at the flanking region of pTZ57R multiple cloning site. Conclusion: The highest advantage of this cost-benefit approach is to produce different types of molecular weight markers by using an effective and short protocol
Volume 70, Issue 4 (7-2012)
Background: Use of certain antipsychotic drugs has severe effects on fertility in males. Hypothalamus and hypophysial impressions and changes in plasma hormones concentration like prolactin, LH and FSH can affect sperm production. In this study, we investigated the effects of sulpiride on sperm quality, maturation and DNA damage.
Methods: Twenty for adult male mice (age: 6-8 weeks) were divided into three groups. The treatment group received 40 mg/kg sulpiride solution and the control sham group was given carrier of the drug intraperitoneally (IP) daily for 45 days but the control group received nothing. Finally, all the mice were sacrificed by cervical dislocation and their cauda epididymis were removed surgically. The excised specimens were placed in 1 ml HTF medium and incubated for 30 min in CO2 incubator to allow the spermatozoa to swim out. Later, sperm count, motility and viability were analyzed. Additionally, sperm chromatin quality and DNA integrity were assessed by aniline blue and acridine orange staining.
Results: Significant decrease in sperm motility and count were observed in the treatment group while the number of abnormal sperm increased as compared with the other two groups. Sperm viability and DNA maturation showed significant reduction and the rate of DNA damage increased in comparison with the control sham and the control groups (P<0.05).
Conclusion: The study showed that sulpiride has negative effects on sperm parameters in treated animals and in some cases it could cause secondary infertility.
Volume 73, Issue 3 (6-2015)
In situ hybridization (ISH) is a method that uses labeled complementary single strand DNA or RNA to localize specific DNA or RNA sequences in an intact cell or in a fixed tissue section. The main steps of ISH consist of: probe selection, tissue or sample preparation, pre-hybridization treatment, hybridization and washing, detection and control procedure. Probe selection is one of the important aspects of successful hybridization. ISH sensitivity and specificity can be influenced by: probe construct, efficiency of labeling, percentage of GC, probe length and signal detection systems. Different methods such as nick translation, random priming, end tailing and T4 DNA polymerase replacement are used for probe generation. Both radioactive and non-radioactive labels can be used in order to probe labeling. Nucleic acid maintenance in samples, prevention of morphological changes of samples and probe penetration into tissue section are the main aims of sample preparation step. Then, a small amount of solution containing probe, is added on slides containing tissue sections for hybridization process, then slides are incubated overnight. Next day, washes are carried out to remove the probes which are not bound to target DNA or RNA. Finally, in order to be sure that the observed labeling is specific to the target sequence, using several control procedures is very important. Various techniques based on ISH consist of: Fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), genomic in situ hybridization (GISH), comparative genomic hybridization (CGH), spectral karyotyping (SKY) and multiplex fluorescence in situ hybridization (MFISH). One of the most common techniques of ISH is fluorescence in situ hybridization. FISH can be used to: 1) detect small deletions and duplications that are not visible using microscope analysis, 2) detect how many chromosomes of a certain type are present in each cell and 3) confirm rearrangements that are suspected after microscope analysis. In this technique different fluorescent labels are attached to the probes. In this review article ISH, its different types, their application, advantages and disadvantages have been considered.
Volume 74, Issue 8 (11-2016)
Stable molecular changes during cell division without any change in the sequence of DNA molecules is known as epigenetic. Molecular mechanisms involved in this process, including histone modifications, methylation of DNA, protein complex and RNA antisense. Cancer genome changes happen through a combination of DNA hypermethylation, long-term epigenetic silencing with heterozygosis loss and genomic regions loss. Different combinations of N-terminal’s changes cooperate with histone variants with a specific role in gene regulation. It have led to load a setting histone that determine transcription potential of a particular gene or genomic regions. DNA methylation analysis in genome region using methylation-specific digital karyotyping of normal breast tissue detect gene expression patterns and DNA specific methylation can be found in breast carcinoma too more than 100 genes in breast tumors or cell lines of breast cancer are reported hypermethylated. Important of DNA methylation on cancer has been concentrated CpG islands hypermethylation. Most of the techniques are able to identify hypermethylated areas. Often, methylated genes play important role in cell cycle regulation, apoptosis, metastasis and tissue invasion, angiogenesis and hormonal signaling. Cyclin D2 (CCND2) gene is an important regulator of cell cycle and increased of expression inhibits the transition from G1 to S cell cycle. This gene is frequently methylated in breast cancer and has been proposed as the first event. Other cell cycle regulator is p16ink4A / CDKN2A that methylated in a large number of human cancers, including breast cancer. Another regulator of the proliferation of breast cancer that methylated is tumor suppressor RAR-β cancer that has been found in lobular and ductal carcinoma. Recent studies have showed the role of epigenetic silencing in the pathogenesis of breast cancer in which tumor suppressor genes have been changed by acetylation and DNA deacetylation. Histone deacetylase inhibitors have different roles in cancer cells and could show the ways of new treatment for breast cancer. In this review, various aspects of breast cancer epigenetics and its applications in diagnosis, prediction and treatment are described.
Volume 74, Issue 8 (11-2016)
Background: Problems of live and inactivated influenza vaccines such as, increasing emerge and re-emerge viruses with high human mortality, current epidemics of influenza and direct transmission of avian viruses to human, affect the vaccination program. DNA vaccines as third generation of vaccines is specially considered for control of influenza in human and poultry. The main advantage of these vaccines is humoral and cellular immune responses and broad spectrum of using these vaccines for control of circulating strains of influenza. In this study the conserved fragment of HA2 to form of DNA vaccine was designed to induce immunity against influenza viruses and its heterologous protective immunity against these viruses was evaluated.
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Methods: The experimental study was performed in Razi Vaccine and Serum Research Institute from December 2014 to July 2015 in Iran. The HA2 was cloned into pcDNA3.1 to assess the HA2 DNA vaccine and mice were immunized with the generated constructs in a DNA prime-DNA boost regimen in 4 groups. The humoral immune responses were analyzed at defined intervals by VN tests. The safety of the vaccine was evaluated by daily inspection and histopathological examination. For evaluation of cellular immunity, proliferation assay was used. Results: The antibody titre and cellular immunity of immunized mice was significantly higher than control group for two serotypes and the highest responses was in the group with two-time boosting (P<0.01). There were no any local, general and histopathology reactions in immunized mice. Conclusion: The HA2 DNA vaccine significantly enhanced circulatory antibody responses and cellular immunity against influenza current serotypes. This study showed the highest immune responses were in the group that immunized with HA2 in prime and two boosts. Besides that, this construct did not have any local and general reaction and any side effects in treated mice. So, this construct was introduced as candidate for control of influenza virus serotypes. |
Volume 75, Issue 6 (9-2017)
Methods: In this case-control study, semen samples were collected from 35 infertile men with varicocele (grade II & III) and 20 fertile men referring to Isfahan Fertility and Infertility Center from January 2016 to September 2016. Ejaculated semen was obtained by masturbation into a sterile plastic container after 3-5 days of abstinence and was allowed to liquefy at room temperature. Briefly, sperm concentration, motility and morphology were evaluated using a sperm chamber (Sperm meter; Sperm Processor, Aurangabad, India), Computer Assisted Semen Analysis (CASA, Video Test, ltd: version Sperm 2.1, Russia) and Papanicolaou staining, respectively. In addition, DNA fragmentation, expression of PAWP at RNA and protein levels were assessed by real-time PCR, and Western blot technique, respectively. Microsoft Excel 2013 (Microsoft Corp., Redmond, WA, USA) and Package for the Social Studies were used to analyze data. We used independent-samples t-test to compare the mean value between different groups. Differences with values of P<0.05 were considered as statistically significant.
Results: Mean of semen volume, sperm concentration, percentage of sperm motility, and expression of PAWP at both RNA (P=0.0001) and protein (P=0.03) levels were significantly lower in infertile men with varicocele in comparison with fertile men. In addition, mean percentage of sperm abnormal morphology and sperm DNA fragmentation were significantly higher in infertile men with varicocele in comparison with fertile men (P<0.05).
Conclusion: Expression of PAWP as a protein involved in spermatogenesis and fertilization process, has decreased in infertile men with varicocele. In addition, sperm DNA integrity was disrupted in these individuals that can be considered as a main etiology of infertility.
Volume 76, Issue 9 (12-2018)
Methods: A systematic review was performed based on search in international databases with no time limit including Scopus, PubMed and Google Scholar. All databases were searched using the standard (English and Persian) keywords. Relevant articles from 1996 to 2018 were collected from search of international databases including Science Direct, Google Scholar, and PubMed using keywords such as “shuttle vectors”, “recombinant plasmids” and “DNA vaccines”.
Results: In this study, a total of 31 full texts were used. A shuttle vector typically contains similar components to replication origins and promoters and can propagate in various hosts. Nowadays, they are used in designing and constructing of new vaccines against infectious diseases including tuberculosis and viral hepatitis. Also, Multi-epitope peptide DNA vaccines are effective against some viruses and they are potentially effective against some bacteria such as Helicobacter pylori.
Conclusion: Shuttle vectors as a powerful genetic engineering tool have a high ability to study the mechanisms of pathogenic microorganisms and make new vaccines such as DNA vaccines and multi-epitope vaccines. The hope is that such multi-epitope DNA vaccines might induce immunity against multiple antigenic targets, multiple strain variants, and/or even multiple pathogens. However, the ability of DNA vaccination to co-deliver a series of antibody and/or CD4 T cell epitopes remains largely unexplored.
Volume 76, Issue 10 (1-2019)
Methods: This case-control study was carried out on the women with breast cancer and healthy women located in Markazi province at Arak University Research, Iran, from October 2016 to March 2017. In the present study, the association of FokI polymorphisms and the risk of breast cancer was assessed by Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. In this method, genomic DNA was extracted from blood samples using the kit procedure. Then, PCR was performed and the SNP-containing DNA amplicons were subjected to digestion of enzymes. Following digestion, each sample was immediately analyzed by 3% agarose gel electrophoresis. Statistical analysis was done using SPSS and SNP Analyzer softwares and the final results were determined.
Results: No statistically significant difference was observed between the two groups of patients and controls for three genotypes the site rs1799794 (P=0.435). Genotype AG (P=0.384, OR=0.614, CI=95%, 0.205-1.840) and GG (P=0.867, OR=0.911, CI=95%; 0.308-2.699) had no significant associations with risk of breast cancer.
Conclusion: There was no significant association between FokI polymorphisms of the XRCC3 and risk of susceptibility to breast cancer, which was in accordance to some researchers. FokI polymorphisms of XRCC3 gene cannot be used as a biomarker in clinical predictive studies in relation to risk of breast cancer.
Volume 80, Issue 7 (10-2022)
Methods: In a case/control study, three groups were selected consisting of 32 opium abusers, 24 heroin addicts and 25 healthy individuals. The amount of 5 ml of whole blood was collected from each individual who participated in the study and stored at -20 centigrade. The sample collection was performed from November 2018 to February 2020. Case groups were recruited from the Methadone maintenance therapy center. Contro group had no history of drug use and cigarette smoking. DNA was extracted from the whole blood samples using the salting out method. The DNA from a mitochondrial gene, dehydrogenase subunit1 (-ND1 gene) and a nuclear gene, human globulin (HGB gene), were quantified by a real-time PCR-based method to measure the relative mtDNA copy number of each group number.
Results: There was no significant difference in demographic characterization between the three study groups, opium abusers, heroin addicts and healthy individuals. We found that opium users had a higher mean of mtDNA copy number than those in the healthy control group (P=0.11). Heroin addicts had also higher mean of mtDNA copy number than those in healthy group (P=0.21). The mean mtDNA copy number in opium abusers was higher than that in heroin addicts (P=0.22), although the difference was not statistically significant.
Conclusion: The results of this study indicated that mtDNA copy number increased in a group of opiate abusers. Considering that alteration of mtDNA copy number is associated with increased susceptibility to several diseases including cancer, further research on mtDNA copy number with a high number of volunteers of opiate addicts may clear the effect of opiate abuse on the human genome.
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