Highly Sensitive Assay of Methyltransferase Activity Based on an Autonomous Concatenated DNA Circuit.

Methyltransferase-involved DNA methylation is one of the most important epigenetic processes, making the ultrasensitive MTase assay highly desirable in clinical diagnosis as well as biomedical research. Traditional single-stage amplification means often achieve linear amplification that might not fulfill the increasing demands for detecting trace amount of target. It is desirable to construct multistage cascaded amplifiers that allow for enhanced signal amplifications. Herein, a powerful nonenzymatic MTase-sensing platform is successfully engineered based on a two-layered DNA circuit, in which the upstream catalytic hairpin assembly (CHA) circuit successively generates DNA product that could be used to activate the downstream hybridization chain reaction (HCR) circuit, resulting in the generation of a dramatically amplified fluorescence signal. In the absence of M.SssI MTase, HpaII endonuclease could specifically recognize the auxiliary hairpin substrate and then catalytically cleave the corresponding recognition site, releasing a DNA fragment that triggers the CHA-HCR-mediated FRET transduction. Yet the M.SssI-methylated hairpin substrate could not be cleaved by HpaII enzyme, and thus prohibits the CHA-HCR-mediated FRET generation, providing a substantial signal difference with that of MTase-absent system. Taking advantage of the high specificity of multiple-guaranteed recognitions of MTase/endonuclease and the synergistic amplification features of concatenated CHA-HCR circuit, this method enables an ultrasensitive detection of MTase and its inhibitors in serum and E. coli cells. Furthermore, the rationally assembled CHA-HCR also allows for probing other different biotransformations through a facile design of the corresponding substrates. It is anticipated that the infinite layer of multilayered DNA circuit could further improve the signal gain of the system for accurately detecting other important biomarkers, and thus holds great promise for cancerous treatment and biomedical research.

Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase (hTERT) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme MspI for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency.

Identification of medically important Candida species by polymerase chain reaction-restriction fragment length polymorphism analysis of the rDNA ITS1 and ITS2 regions.

AIM: We aimed to identify the distribution of species in candidal strains isolated from clinical samples and restriction fragment length polymorphism (RFLP) method based on Msp I and Bln I restrictive enzyme cuts of polymerase chain reaction (PCR) products after the amplification of ITS1 and ITS2 regions of rDNA genotypically. MATERIALS AND METHODS: One hundred and fifty candidal strains isolated from various clinical samples were studies/ included. Phenotypic species assessment was performed using automated VITEK-2 system and kit used with the biochemical tests. Common genomic region amplification peculiar to candidal strains was carried out using ITS1 and ITS2 primer pairs. After the amplification, PCR products were cut with Msp I and Bln I restriction enzymes for species identification. RESULTS: The majority of Candida isolates were isolated from urine (78.6%) while other isolates were composed of strains isolated from swab, wound, blood and other samples by 11.3%, 3.3%, 2% and 4.7%, respectively. The result of RFLP analysis carried out with Msp I and Bln I restriction enzymes showed that candidal strains were Candida albicans by 45.3%, Candida glabrata by 19.3%, Candida tropicalis by 14.6%, Candida parapsilosis by 5.3%, Candida krusei by 5.3%, Candida lusitaniae by 0.6% and other candidal strains by 9.3%. CONCLUSION: When the ability to identify Candida to species level of phenotypic and PCR-RFLP methods was assessed, a great difference was found between these two methods. It may be argued that Msp I and Bln I restriction enzyme fragments can be used in the identification of medically important Candida species. Further studies are needed to develop this kind of restriction profile to be used in the identification of candidal strains.

Association of CYP1A1 (cytochrome P450) MspI polymorphism in women with endometriosis.

Endometriosis is a disease that affects 10 to 15% of the women of reproductive age. It is characterized by the presence of endometrial-like tissues outside of the uterus. Some definitions claim that the functional ectopic tissue is sensitive to the action of hormones. Severity of endometriosis is defined according to a system proposed by the American Society for Reproductive Medicine, which is based on laparoscopic findings. A large number of genetic polymorphisms has been reported for CYP1A1, the gene that is responsible for enzymes involved in stage I detoxification of xenobiotics; this gene is located at 15q22-24, and encodes an isoenzyme that catalyzes the oxidation of polycyclic aromatic hydrocarbons present in phenolic compounds and epoxides. The aim of this study was to analyze the frequency of the MspI polymorphism and its relation to endometriosis. We obtained peripheral blood samples from 52 women with endometriosis (confirmed by laparoscopy) as well as 42 women without endometriosis (control group). In the case group, the women were between 25 and 35 years of age; the age range was between 25 and 57 years old in the control group. Molecular analysis was performed by polymerase chain reaction. We found a significant association (P = 0.039) between the polymorphic allele m1 and endometriosis (32.70%). In conclusion, this study showed that the m1 polymorphism is associated with endometriosis, and that W1/m1 and m1/m1 polymorphisms are more frequently observed in patients with infertility and severe endometriosis.

Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB.

We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes.

Effect of Genetic Polymorphisms in GH/Hpa II and MSTN / Dra I Loci on Body Weight in Friesian Bull Calves.

OBJECTIVE: The GH and MSTN gene polymorphisms and their association with body weight were declared in a population of 100 Friesian bull calves. MATERIALS AND METHODS: For DNA extraction, collection of blood samples was carried out from the studied animals. The PCR for GH and MSTN genes yielded fragments of 329 and 1346 bp, respectively. RESULTS: The PCR-HpaII digestion of 329 bp of GH gene revealed three genotypes: AA genotype possess undigested fragment (329 bp), AB genotype has three fragments (329, 224 and 105 bp) and BB genotype has two fragments (224 and 105 bp). The GH genotypes incidence and alleles frequency were calculated. For the 100 Friesian bull calves, genotypic frequencies for the AA, AB and BB genotypes were 0.1, 0.78 and 0.12, respectively and the allele frequencies for A and B allele frequencies were 0.49 and 0.51. Statistical analysis revealed that there was a significant effect of GH genotypes on body weight. The AB genotype possessed higher body weight than the other 2 genotypes. Regarding MSTN gene, PCR-DraI digestion of 1346 bp fragment was monomorphic; where it yielded four fragments (505, 427, 321 and 93 bp) in all animals under study. CONCLUSION: The outcome of this study is that it highlights the effectiveness of GH/HpaII locus as candidate marker for body weight in cattle rather than MSTN/DraI.

Evaluation of DNA methyltransferase activity and inhibition via chiroplasmonic assemblies of gold nanoparticles.

Circular dichroism spectroscopy has been explored for detection of methyltransferase activity and inhibition based on DNA-induced chiroplasmonic assemblies of gold nanoparticles and endonuclease HpaII. Good accuracy, precision and sensitivity are obtained in complex matrices such as human serum samples, which is significant for clinical diagnosis and drug development.

Label-free electrochemical detection of methyltransferase activity and inhibitor screening based on endonuclease HpaII and the deposition of polyaniline.

Detection of DNA methylation and methyltransferase (MTase) activity are important in determining human cancer because aberrant methylation was linked to cancer initiation and progression. In this work, we proposed an electrochemical method for sensitive detection of DNA methylation and MTase activity based on methylation sensitive restriction endonuclease HpaII and the deposition of polyaniline (PANI) catalyzed by HRP-mimicking DNAzyme. In the presence of methylated DNA, HRP-mimicking DNAzyme catalyzed the polymerization of aniline on the dsDNA template, producing huge DPV current. In the presence of non-methylated DNA, dsDNA are cleaved and digested by HpaII and exonuclease III, as a result, no PANI are deposited. This method can be used to determine DNA methylation at the site of CpG. It exhibits a wide linear response toward M.SssI MTase activity in the range of 0.5-0.6 U mL(-1) with the detection limit of 0.12 U mL(-1). G-rich DNA forms HRP mimicking DNAzyme, which avoids complex labeling procedures and is robust. The method is simple, reliable, sensitive and specific, which has been successfully applied in human serum samples and been used to screen the inhibitors. Thus, the proposed method may be a potential and powerful tool for clinical diagnosis and drug development in the future.

Association of CYP1A1 MspI polymorphism in the esophageal cancer risk: a meta-analysis in the Chinese population.

BACKGROUND: Although many epidemiologic studies have investigated the CYP1A1 MspI gene polymorphisms and their associations with esophageal cancer (EC), definite conclusions cannot be drawn. To clarify the effects of CYP1A1 MspI polymorphisms on the risk of EC, a meta-analysis was performed in Chinese population. METHODS: Related studies were identified from PubMed, Springer Link, Ovid, Chinese Wanfang Data Knowledge Service Platform, Chinese National Knowledge Infrastructure (CNKI), and Chinese Biology Medicine (CBM) till October 2014. Pooled ORs and 95% CIs were used to assess the strength of the associations. RESULTS: A total of 13 studies including 1,519 EC cases and 1,962 controls were involved in this meta-analysis. Overall, significant association was found between CYP1A1 MspI polymorphism and EC risk when all studies in the Chinese population pooled into this meta-analysis (C vs. T: OR = 1.25, 95% CI = 1.04 to 1.51; CC + CT vs. TT: OR = 1.35, 95% CI = 1.06 to 1.72; CC vs. TT + CT: OR = 1.35, 95% CI = 1.03 to 1.76). When we performed stratified analyses by geographical locations, histopathology type, and source of control, significantly increased risks were found in North China (C vs. T: OR = 1.38, 95% CI = 1.12 to 1.70; CC vs. TT: OR = 1.72, 95% CI = 1.16 to 2.56; CC + CT vs. TT: OR = 1.52, 95% CI = 1.14 to 2.02; CC vs. TT + CT: OR = 1.55, 95% CI = 1.17 to 2.06), in the population-based studies (C vs. T: OR = 1.22, 95% CI = 1.05 to 1.42; CC vs. TT: OR = 1.38, 95% CI = 1.02 to 1.88; CC + CT vs. TT: OR = 1.36, 95% CI = 1.10 to 1.69; CC vs. TT + CT: OR = 1.43, 95% CI = 1.13 to 1.81) and ESCC (C vs. T: OR = 1.17, 95% CI = 1.04 to 1.32; CC + CT vs. TT: OR = 1.28, 95% CI = 1.08 to 1.52). CONCLUSIONS: This meta-analysis provides the evidence that CYP1A1 MspI polymorphism may contribute to the EC development in the Chinese population.

A fluorescence method for detection of DNA and DNA methylation based on graphene oxide and restriction endonuclease HpaII.

DNA methylation plays an important role in many biological events and is associated with various diseases. Most traditional methods for detection of DNA methylation are based on the complex and expensive bisulfite method. In this paper, we report a novel fluorescence method to detect DNA and DNA methylation based on graphene oxide (GO) and restriction endonuclease HpaII. The skillfully designed probe DNA labeled with 5-carboxyfluorescein (FAM) and optimized GO concentration keep the probe/target DNA still adsorbed on the GO. After the cleavage action of HpaII the labeled FAM is released from the GO surface and its fluorescence recovers, which could be used to detect DNA in the linear range of 50 pM-50 nM with a detection limit of 43 pM. DNA methylation induced by transmethylase (Mtase) or other chemical reagents prevents HpaII from recognizing and cleaving the specific site; as a result, fluorescence cannot recover. The fluorescence recovery efficiency is closely related to the DNA methylation level, which can be used to detect DNA methylation by comparing it with the fluorescence in the presence of intact target DNA. The method for detection of DNA and DNA methylation is simple, reliable and accurate.

Single base resolution analysis of 5-methylcytosine and 5-hydroxymethylcytosine by RRBS and TAB-RRBS.

Sodium bisulfite-assisted deamination of cytosine forms the basis for conducting single base resolution analysis of 5-methylcytosine in DNA. The TET family of proteins represents a group of enzymes that can oxidize 5-methylcytosine to 5-hydroxymethylcytosine. A modification of the bisulfite-based DNA methylation mapping technique employs TET1-mediated oxidation of 5-methylcytosine (TET-assisted bisulfite sequencing) for single base analysis of 5-hydroxymethylcytosine. Whole genome analysis of cytosine modifications with bisulfite sequencing techniques still is challenging and expensive. Reduced representation bisulfite sequencing (RRBS) has been used to limit the complexity of the analysis to mostly CpG-rich genomic fragments flanked by restriction enzyme cleavage sites, for example MspI (5'CCGG). In this chapter, we describe detailed methods used in our laboratory for analysis of 5-methylcytosine and 5-hydroxymethylcytosine combined (RRBS) and for specific analysis of 5-hydroxymethylcytosine (TAB-RRBS).

Ultrasensitive electrochemical biosensing for DNA using quantum dots combined with restriction endonuclease.

A universal and sensitive electrochemical biosensing platform for the detection and identification of DNA using CdSe quantum dots (CdSe QDs) as signal markers was designed. The detection mechanism was based on the specific recognition of MspI endonuclease combined with the signal amplification of gold nanoparticles (AuNPs). MspI endonuclease could recognize its specific sequence in the double-strand DNA (dsDNA) and cleave the dsDNA fragments linked with CdSe QDs from the electrode. The remaining attached CdSe QDs can be easily read out by square-wave voltammetry using an electrodeposited bismuth (Bi) film-modified glass carbon electrode. The concentrations of target DNA could be simultaneously detected by the signal of metal markers. Using mycobacterium tuberculosis (Mtb) DNA as a model, under the optimal conditions, the proposed biosensor could detect Mtb DNA down to 8.7 × 10(-15) M with a linear range of 5 orders of magnitude (from 1.0 × 10(-14) to 1.0 × 10(-9) M) and discriminate mismatched DNA with high selectivity. This strategy presented a universal and convenient biosensing platform for DNA assay, and its satisfactory performances make it a potential candidate for the early diagnosis of gene-related diseases.

[MspI-POLYMORPHISM IN FOURTH INTRON OF THE GROWTH HORMONE GENE IN CHICKEN POPULATIONS OF DIFFERENT BREEDS. ANALYSIS OF THE CAUSES OF ADDITIONAL RESTRICTION PATTERN ORIGIN].

The MspI-polymorphism in the fourth intron of the growth hormone gene in populations of White Plymouth Rock, Poltava Clay, Rhode Island Red and Borkovskaya Barvistaya chicken breeds was studied. It is shown that in all examined chicken populations the growth hormone gene is polymorphic. It was found that the presence of "additional" phenotype (restriction pattern) is not associated with duplication of the growth hormone gene. The possibility of formation of heteroduplex DNA of two different types in the course of amplification of heterozygous samples B/C, containing the site 'CCGG', which leads to formation the additional DNA fragment which do not contain the site 'CCGG', was described. The nucleotide sequences of alleles A, B, C and "additional" fragment is described. Frequencies of alleles A, B and C in chicken population of White Plymouth Rock breed were 0.56; 0.16 and 0.28; Poltava Clay--0.10; 0.07 and 0.83; Rhode Island Red--0.27; 0.31 and 0.42; Borkovskaya Barvistaya--0.75; 0.08 and 0.17 respectively.

Label-free fluorescence detection of DNA methylation and methyltransferase activity based on restriction endonuclease HpaII and exonuclease III.

Strategies to detect the methylation of site specific DNA and assay of M.SssI methyltransferase (M.SssI MTase) activity are important in determining human cancers due to aberrant methylation linked to cancer initiation and progression. Herein, we report a label-free fluorescence detection method for DNA methylation and MTase activity based on restriction endonuclease HpaII and exonuclease III (Exo III). A label-free probe DNA was designed, which hybridized with target DNA (one 32-mer DNA from the exon 8 promoter region of the Homo sapiens p53 gene) to form double stranded DNA (dsDNA). Upon the cleavage action of HpaII and degradation reaction of Exo III, dsDNA changed to single stranded DNA (ssDNA) and the fluorescence intensity of thiazole orange (TO) is weak. After the resulting dsDNA was methylated by M.SssI MTase, the action of HpaII and Exo III was prevented, then TO intercalates into the dsDNA and emits strong fluorescence. This method can determine DNA methylation at the site of CpG and distinguish a one-base mismatched target sequence. The fluorescence intensity has a linear relationship with M.SssI MTase activities in the range of 1-10 U mL(-1) with a detection limit of 0.16 U mL(-1) in terms of 3 times deviation of the blank sample. The methylation of DNA by a hydroxyl radical triggered by DMSO and CH3CHO was also measured. These results show that the proposed method can specifically and selectively detect DNA methylation and M.SssI MTase activity. Human serum has no obvious effects on the assay performance, indicating that the method has great potential for further application in complex samples.

Combined effects of CYP1A1 MspI and GSTM1 genetic polymorphisms on risk of lung cancer: an updated meta-analysis.

Genetic polymorphisms of cytochrome P450 1A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1) genes might contribute to the variability in individual susceptibility to lung cancer, but the reported results from individual studies are not always consistent. We therefore conducted a meta-analysis to systematically estimate the associations between polymorphisms of these two genes and risk of lung cancer. Twenty-one studies with 8,926 subjects were finally enrolled into this study. Meta-analysis was performed by RevMan 5.2. Odds ratio (OR) and its 95 % confidence interval (CI) were calculated to evaluate the susceptibility to lung cancer. Compared with the wild-type homozygous genotype, significantly elevated risk of lung cancer were associated with variant CYP1A1 MspI (m1/m2 + m2/m2 vs. m1/m1: OR = 1.27, 95 % CI = 1.12-1.43, P < 0.001) and deletion of GSTM1 (null vs. present: OR = 1.26, 95 % CI = 1.13-1.40, P < 0.001). Both the two genetic polymorphisms were independently associated with the risk of lung cancer. The pooled OR of lung cancer for population with both CYP1A1 MspI and GSTM1 mutations (MspI m1/m2 or m2/m2 and GSTM1 null) was 1.62 (95 % CI 1.27-2.07, P < 0.001) when compared with those without any of the above mutations, which is higher than single genetic polymorphism. In the stratified analysis, significantly higher risks of lung cancer associated with above genetic polymorphisms were found only in Asian population. This meta-analysis suggests that the CYP1A1 MspI and GSTM1 polymorphisms correlate with increased lung cancer susceptibility independently, and that there is an interaction between the two genes. However, the associations vary in different ethnic populations.

Association between cytochrome P450 1A1 MspI polymorphism and endometrial cancer risk: a meta-analysis.

Many studies proposed that cytochrome P450 1A1 (CYP1A1) MspI polymorphism may be associated with endometrial cancer risk, but the findings from previous studies reported conflicting results. A meta-analysis of all relevant studies was performed to get a comprehensive assessment of the association between CYP1A1 MspI polymorphism and endometrial cancer risk. Eligible studies were searched in PubMed and China National Knowledge Infrastructure databases. The pooled odds ratios (ORs) with the corresponding 95 % confidence intervals (95 % CIs) were calculated to evaluate the association. Twelve studies with a total of 2,111 cases and 2,894 controls were finally included into the meta-analysis. Overall, meta-analysis of a total of 12 studies showed that there was no obvious association between CYP1A1 MspI polymorphism and endometrial cancer risk (ORC vs. T = 0.97, 95 % CI 0.77-1.22, P OR = 0.808; ORCC vs. TT = 1.00, 95 % CI 0.57-1.76, P OR = 0.994; ORCC vs. TT/TC = 0.88, 95 % CI 0.65-1.20, P OR = 0.425; ORCC/TC vs. TT = 0.98, 95 % CI 0.74-1.29, P OR = 0.861). Subgroup analyses by ethnicity further showed that there was no obvious association between CYP1A1 MspI polymorphism and endometrial cancer risk in both Caucasians and Asians. There was no obvious risk of publication bias. Therefore, the meta-analysis suggests that CYP1A1 MspI polymorphism is not associated with endometrial cancer risk.

DNA assembly and enzymatic cutting in solutions: a gold nanoparticle based SERS detection strategy.

The ability to monitor biomolecular recognition such as DNA hybridization and enzymatic reactivity in solutions with high sensitivity is important for developing effective bioassay strategies. Surface enhanced Raman scattering (SERS) based on use of solid substrates to produce the SERS effect for the detection often requires substrate preparation which is ineffective for rapid monitoring. This report describes a new strategy exploiting a gold nanoparticle (AuNP) based interparticle "hot-spot" for SERS monitoring of DNA mediated assembly and enzyme induced cleavage of the assembly in solution phase. The DNAs consist of two different complementary DNA strands with a thiol modification for attachment to AuNPs of selected sizes. In a solution containing AuNPs conjugated with one of the single-stranded (ss) DNA and other AuNPs labeled with a Raman reporter molecule, 4-mercaptobenzoic acid (MBA), the introduction of the complementary DNA strand leads to a linkage of the two types of AuNPs, producing double-stranded (ds) DNA-AuNP assembly (ds-DNA-AuNPs) with an interparticle "hot-spot" for SERS detection of the diagnostic bands of the reporter. Upon introducing a restriction enzyme (e.g. MspI) into the ds-DNA-AuNP assembly solution, the removal of the interparticle "hot-spot" due to restriction enzyme cleavage of the ds-DNA leads to a decrease of the SERS signals. While the detailed cleavage process may depend on the reaction time and the amount of enzyme, the viability of using gold nanoparticle "hot-spot" based SERS monitoring of DNA assembly and enzyme cleavage is clearly demonstrated, which has important implications for developing new strategies for bioassays.

Combined methylation mapping of 5mC and 5hmC during early embryonic stages in bovine.

BACKGROUND: It was recently established that changes in methylation during development are dynamic and involve both methylation and demethylation processes. Yet, which genomic sites are changing and what are the contributions of methylation (5mC) and hydroxymethylation (5hmC) to this epigenetic remodeling is still unknown. When studying early development, options for methylation profiling are limited by the unavailability of sufficient DNA material from these scarce samples and limitations are aggravated in non-model species due to the lack of technological platforms. We therefore sought to obtain a representation of differentially 5mC or 5hmC loci during bovine early embryo stages through the use of three complementary methods, based on selective methyl-sensitive restriction and enrichment by ligation-mediated PCR or on subtractive hybridization. Using these strategies, libraries of putative methylation and hydroxymethylated sites were generated from Day-7 and Day-12 bovine embryos. RESULTS: Over 1.2 million sequencing reads were analyzed, resulting in 151,501 contigs, of which 69,136 were uniquely positioned on the genome. A total of 101,461 putative methylated sites were identified. The output of the three methods differed in genomic coverage as well as in the nature of the identified sites. The classical MspI/HpaII combination of restriction enzymes targeted CpG islands whereas the other methods covered 5mC and 5hmC sites outside of these regions. Data analysis suggests a transition of these methylation marks between Day-7 and Day-12 embryos in specific classes of repeat-containing elements. CONCLUSIONS: Our combined strategy offers a genomic map of the distribution of cytosine methylation/hydroxymethylation during early bovine embryo development. These results support the hypothesis of a regulatory phase of hypomethylation in repeat sequences during early embryogenesis.

No association between MspI allele of the ADRA2A polymorphism and ADHD: meta-analysis of family-based studies.

There is evidence for a genetic contribution to attention-deficit hyperactivity disorder (ADHD), although no candidate genes have attained genome-wide significance to date. Given that the noradrenergic system has been implicated in ADHD, the gene for the α2-adrenergic receptor (ADRA2A) has been hypothesized to contribute to the pathogenesis of ADHD. The present investigation reports results from a meta-analysis of family-based studies that did not find a significant association between the MspI polymorphism of the ADRA2A gene and ADHD.

Molecular identification and distribution profile of Candida species isolated from Iranian patients.

A total of 855 yeast strains isolated from different clinical specimens, mainly nail (42%) and vulva-vagina (25%) were identified by a set of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Genomic DNA was extracted from fresh colonies using Whatman FTA Card technology. PCR assays were performed on the complete ribosomal DNA internal transcribed spacer (rDNA-ITS) region for all isolates and species identification was carried out through their specific electrophoretic profiles after digestion with the enzyme MspI. Those isolates suspected as Candida parapsilosis group were then subjected to amplification of the secondary alcohol dehydrogenase (SADH) gene and restriction digestion with NlaIII enzyme. In total, 71.1% of the strains were obtained from females and 28.9% from males. The age group of 31-40 years consisted of the highest frequency of patients with candidiasis. Candida albicans was the predominant species (58.6%) followed by C. parapsilosis (11.0%), C. glabrata (8.3%), C. tropicalis (7.0%), C. kefyr (5.8%), C. krusei (4.4%), C. orthopsilosis (2.1%), and C. guilliermondii (0.6%). A few strains of C. lusitaniae, C. rugosa, C. intermedia, C. inconspicua, C. neoformans and S. cerevisiae were isolated. We could not identify 8 (0.9%) isolates. Candida albicans remains the most frequently species isolated from Iranian patients; however, the number of non-C. albicans Candida species looks to be increasing. The simple and reliable PCR-RFLP system used in the study has the potential to identify most clinically isolated yeasts.